The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
Paul P Van Veldhoven - One of the best experts on this subject based on the ideXlab platform.
-
subcellular study of sphingoid base phosphorylation in rat tissues evidence for multiple sphingosine kinases
Biochimica et Biophysica Acta, 2001Co-Authors: Sofie Gijsbers, Gerd Van Der Hoeven, Paul P Van VeldhovenAbstract:Abstract The enzymatic phosphorylation of sphingoid bases was analysed in rat tissues, using d -erythro-[4,5-3H]Sphinganine as substrate. After optimisation of the assay, taking care to block sphingosine-phosphate lyase and sphingosine phosphatase, highest ATP-dependent kinase activities were present in testis, followed by kidney, and intestinal mucosa. Approximately two thirds of the kidney activity were membrane bound, the remaining being cytosolic. Classical cell fractionation studies of kidney and liver did not allow to identify unequivocally the subcellular site of the membrane bound kinase. Separation of a particulate fraction from kidney homogenates by Percoll gradient and sucrose density gradient centrifugation revealed that kinase activities are associated with vesicles derived from the endoplasmic reticulum and the plasma membrane. Based on indirect data, such as the effect of detergents and divalent ions, the cytosolic and both membrane bound activities appear to reside in different proteins. N,N-Dimethylsphingenine was inhibitory to all three different kinases, which were mainly active towards the d -erythro isomers of sphingenine and Sphinganine.
-
Do sphingoid bases interact with the peroxisome proliferator activated receptor α (PPAR-α)?
Cellular Signalling, 2000Co-Authors: Paul P Van Veldhoven, Guy P. Mannaerts, Peter Declercq, Myriam BaesAbstract:Abstract In a search for possible endogenous ligands of nuclear receptors that are activated by peroxisome proliferators (PPARs), a solid phase binding assay was developed employing recombinant mouse PPAR-α, containing a myc-epitope, a histidine repeat and a kinase A domain. After in vitro labelling with 32P-γ-ATP, the binding of purified 32P-PPAR-α to a panel of different natural and synthetic lipids, immobilized on silica layers, was evaluated. Autoradiographs of the silica layers revealed binding to two main classes of lipophilic compounds. A first class comprised (poly)unsaturated fatty acids. Compounds belonging to a second class were characterized by the presence of an overall positive charge such as long chain amines, sphingoid bases (sphingenine), and lysoglycosphingolipids (psychosine). PPAR-α did not bind to N-acylated sphingoid bases (ceramides) or to sphingenine phosphorylated at the primary hydroxy group (sphingenine-1-phosphate). The binding of PPAR-α to sphingoid bases might be of interest given the role of PPAR-α and sphingolipids in various cellular processes.
-
identification and subcellular localization of Sphinganine phosphatases in rat liver
Biochemical Journal, 1995Co-Authors: P De Ceuster, Guy P. Mannaerts, Paul P Van VeldhovenAbstract:One of the primary products of [4,5-3H]Sphinganine phosphate, added to fibroblast cultures, is Sphinganine [Van Veldhoven and Mannaerts (1994) Biochem. J. 299, 597-601], implicating the physiological action of (a) hitherto unknown phosphatase(s). We have now further characterized this activity in rat liver. In homogenates, the dephosphorylation appeared to be catalysed by multiple enzymes. A low-affinity system was active at acidic pH, whereas at physiological pH values hydrolysis was carried out by a high-affinity enzyme. The latter was sensitive to Zn2+ and detergents and possessed a pH optimum of 7.5. Upon cell fractionation the major portion of the high-affinity activity was recovered in the nuclear and microsomal fractions. Further separation of the microsomal fraction showed an association predominantly with vesicles derived from the plasma membrane. Likewise, when plasma membranes were prepared from the nuclear fraction, the high-affinity phosphatase co-purified with the plasma membrane markers. From the differential effects of bivalent cations, chelators, water-soluble and amphiphilic phosphate esters, detergents and other compounds, it could be concluded that the plasma membrane-associated Sphinganine-phosphatase activity is not due to alkaline phosphatase, dolichol-phosphatase, the N-ethylmaleimide-insensitive phosphatidate phosphatase or ceramide-phosphatase. The dephosphorylation observed at acidic pH in homogenates appeared also to be enriched in purified plasma membranes and might represent a side-activity of ceramide-phosphatase. We speculate that the high-affinity phosphatase, which is especially active in neuronal tissues, plays a role in the attenuation of bioactive phosphorylated sphingoid bases such as sphingenine phosphate, and propose to name it sphingosine-phosphatase.
-
on the presence of phosphorylated sphingoid bases in rat tissues a mass spectrometric approach
FEBS Letters, 1994Co-Authors: Paul P Van Veldhoven, Guy P. Mannaerts, Patrick De Ceuster, Raoul Rozenberg, Edmond De HoffmannAbstract:A simple and straightforward procedure to analyze phosphorylated sphingoid bases has been developed. After phase separation of lipid extracts under alkaline conditions, the compounds were quantitatively recovered in the aqueous upper phase. Following a clean-up of the aqueous phase on C18-solid phase extraction columns, the amino-group of the bases was derivatized by means of phenylisothiocyanate addition. FAB-MS of the phenylthiocarbamate derivatives of sphingenine- and Sphinganine-phosphate in the negative mode revealed the expected pseudo-molecular ions (M-1) at 513 m/z and 515 m/z, respectively. Moreover, a typical fragmentation pattern, characterized by the loss of the phenylthiocarbamate moiety (m/z = 135), was observed. When applied to rat tissues, the presence of sphingenine-phosphate in brain, kidney and liver could easily be demonstrated. Highest levels, amounting to 5 nmol/g of wet weight, were present in brain.
-
Sphinganine 1 phosphate metabolism in cultured skin fibroblasts evidence for the existence of a sphingosine phosphatase
Biochemical Journal, 1994Co-Authors: Paul P Van Veldhoven, Guy P. MannaertsAbstract:On addition of [4,5-3H]Sphinganine 1-phosphate to human fibroblast monolayers, the label was efficiently removed from the culture medium. In contrast with the reported stability of phosphorylated sphingenine in 3T3 cells [Desai, Zhang, Olivera, Mattie and Spiegel (1992). J. Biol. Chem. 267, 23122-23128] and B16 melanoma cells [Sadahira, Ruan, Hakomuri and Igarashi (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9686-9690], Sphinganine 1-phosphate appeared to be subjected to a fast and extensive metabolism in fibroblasts, the major pathways being cleavage and dephosphorylation. The first of these pathways, catalysed by sphingosine-phosphate lyase, resulted in the formation of labelled palmitaldehyde, which was recovered, mainly after oxidation, in glycerophospholipids in an ester bond. A smaller part of the palmitaldehyde was reduced and incorporated in alk(en)ylphospholipids. Dephosphorylation of spinganine 1-phosphate, a hitherto overlooked pathway catalysed by an unknown phosphatase(s), gave rise to Sphinganine, which was converted by N-acylation into ceramide and then incorporated in spingomyelin and glycosphingolipids.
Alfred H. Merrill - One of the best experts on this subject based on the ideXlab platform.
-
induction of apoptosis by fumonisin b1in ht29 cells is mediated by the accumulation of endogenous free sphingoid bases
Toxicology and Applied Pharmacology, 1998Co-Authors: Eva M Schmelz, Mary Ann Dombrinkkurtzman, Paul C Roberts, Yasunori Kozutsumi, Toshisuke Kawasaki, Alfred H. MerrillAbstract:Fumonisin B1(FB1) and aminopentol (AP1) (which is formed by hydrolysis of FB1) are found in corn contaminated with some strains ofFusarium moniliforme.Incubation of HT29 cells (a human colonic cell line) with FB1or AP1caused a significant reduction in cell number; AP1was less potent, with 50 μM AP1causing the same reduction (ca. 30% after 24 h) as 10 μM FB1. The reduction in cell number reflected increases in DNA fragmentation and the percentage of apoptotic cells. Both FB1and AP1caused the accumulation of Sphinganine (25- and 35-fold by 10 μM FB1and 50 μM AP1, respectively); thus, concentrations of FB1and AP1that caused comparable reductions in cell number were also similar with respect to elevation of Sphinganine, a compound that is growth inhibitory and cytotoxic. Inhibition of the first step of sphingolipid biosynthesis with ISP-1 prevented the elevation in Sphinganine, DNA fragmentation, and apoptosis induced by FB1. Therefore, these effects of FB1on HT29 cells can be attributed to the accumulation of Sphinganine. Since consumption of food contaminated withFusarium moniliforme(Sheldon) exposes colonic cells to these mycotoxins, the possibility that FB1and AP1are toxic for intestinal cellsin vivoshould be evaluated, especially in the light of the recent report (Bhatet al., Clin. Toxicol.35, 249, 1997) describing intestinal disturbances in humans after consumption of moldy corn and sorghum containing fumonisins.
-
changing j774a 1 cells to new medium perturbs multiple signaling pathways including the modulation of protein kinase c by endogenous sphingoid bases
Journal of Biological Chemistry, 1997Co-Authors: Elizabeth R B Smith, Peter L Jones, Jeremy M Boss, Alfred H. MerrillAbstract:Abstract Sphingosine, Sphinganine, and other long-chain (sphingoid) bases are highly bioactive intermediates of sphingolipid metabolism that have diverse effects when added to cells, including the inhibition of protein kinase C (PKC) as evaluated by both enzymatic activity and [3H]phorbol dibutyrate ([3H]PDBu) binding. Nonetheless, changes in endogenous sphingoid bases have not been proven to affect PKC or other signal transduction pathways. We have discovered recently that changing J774A.1 cells to new medium results in up to 10-fold increases in sphingoid bases (Smith, E. R., and Merrill, A. H., Jr. (1995) J. Biol. Chem. 270, 18749-18758); therefore, this system was used to elevate sphingosine and Sphinganine and determine if PKC was affected. Incubation of J774A.1 cells in new medium for 30 min increased the levels of these endogenous sphingoid bases to approximately 0.5 nmol/mg of protein and decreased [3H]PDBu binding by 40-60%. Addition of NH4Cl, which suppresses the change in sphingosine, restored [3H]PDBu binding. Elevation of endogenous Sphinganine by a second method (addition of fumonisin B1, an inhibitor of ceramide synthase) also reduced [3H]PDBu binding; therefore, elevations in sphingosine and Sphinganine can both affect PKC. The elevation in sphingoid bases was also associated with an increase in the amount of PKC-δ (the major PKC isozyme in J774A.1 cells) in the cytosol, as determined by activity assays and immunoblot analyses. Changing the culture medium affected other PKC isozymes, increased cellular levels of diacylglycerol, dihydroceramide, and ceramide, and altered the expression of two genes (the expression of JE was increased, and the induction of MnSOD by TNF-α was potentiated). Thus, changing the culture medium has numerous effects on J774A.1 cells, including the modulation of PKC by endogenous sphingoid bases.
-
differential roles of de novo sphingolipid biosynthesis and turnover in the burst of free sphingosine and Sphinganine and their 1 phosphates and n acyl derivatives that occurs upon changing the medium of cells in culture
Journal of Biological Chemistry, 1995Co-Authors: Elizabeth R B Smith, Alfred H. MerrillAbstract:Abstract Long-chain (sphingoid) bases are highly bioactive intermediates of sphingolipid metabolism, yet relatively little is known about how the amounts of these compounds are regulated. This study used J774A.1 cells to characterize the “burst” of Sphinganine and sphingosine, or the transient increase of up to 10-fold in long-chain base mass, that occurs when cells in culture are changed to fresh medium. The increase in Sphinganine was attributable to de novo sphingolipid biosynthesis because: 1) there is increased incorporation of [3H]serine and [3H]palmitate into Sphinganine; 2) the incorporation of [3H]serine was equivalent to the increase in Sphinganine mass; 3) β-F-alanine, an inhibitor of serine palmitoyltransferase, blocked the Sphinganine burst; 4) the magnitude of the burst depended on the concentration of serine in the medium, which is known to affect long-chain base biosynthesis; and 5) the appearance of Sphinganine was relatively unaffected by lyso-osmotrophic agents (NH4Cl and chloroquine) that blocked sphingolipid hydrolysis in these cells. In contrast, the sphingosine burst arose mainly from turnover of complex sphingolipids because no incorporation of [3H]serine or [3H]palmitate into sphingosine was detected; sphingosine mass was not affected by β-F-alanine or the serine concentration; and, the burst could be followed by the release of sphingosine and ceramide from complex sphingolipids (especially sphingomyelin) in a process that was inhibited by NH4Cl and chloroquine. Additionally, the fate of these long-chain bases differed: Sphinganine was mostly (80-85%) acylated and incorporated into dihydroceramide and complex sphingolipids, whereas most of the sphingosine (70%) was phosphorylated and degraded, with incorporation of the resulting ethanolamine phosphate into phosphatidylethanolamine. Sphinganine, however, could be diverted toward degradation by adding an inhibitor of N-acylation (fumonisin B1). In accounting for the elevation in sphingosine and Sphinganine after cells are changed to new medium, these studies have provided fundamental information about long-chain base metabolism. The existence of differential changes in Sphinganine and sphingosine, as well as their 1-phosphates and N-acyl-derivatives, should be considered when evaluating the roles of sphingolipid metabolites in cell regulation.
-
Liquid chromatographic determination of Sphinganine and sphingosine : use of the free Sphinganine-to-sphingosine ratio as a biomarker for consumption of fumonisins
Journal of AOAC International, 1994Co-Authors: Ronald T Riley, Elaine Wang, Alfred H. MerrillAbstract:Abstract Because the chemical structure of fumonisin B1 (FB1) has several structural features in common with the sphingoid bases, sphingosine and dihydro-sphingosine (Sphinganine), we tested the hypothesis that the fumonisins might alter the normal cellular activity or the metabolism of endogenous free sphingoid bases. FB1 was found to be a potent inhibitor of de novo sphingolipid biosynthesis in vitro, its primary target being Sphinganine N-acyl-transferase. This inhibition resulted in a decrease in the biosynthesis of sphingosine and an accumulation of free Sphinganine, an intermediate in the de novo biosynthetic pathway for complex sphin-golipids. These findings led to the hypothesis that consumption of feed containing fumo|nisins should cause an increase in the ratio of free Sphinganine to free sphingosine in tissues and serum. Data consistent with this hypothesis have been obtained from horses and pigs that consumed feed containing fumonisin-contaminated corn screenings and from rats fed feed supplemented with fumonisin-containing fungal culture materials or pure FBi. Thus, the ratio of free Sphinganine to free sphingosine shows promise as a tissue, urine, or serum marker for animals consuming feed containing fumonisins. The present paper provides a detailed description of the extraction of free sphingoid bases and the liquid chromatographic method we used for determining the relative amounts of free sphingosine and free Sphinganine in serum, urine, and various tissues of animals. Study results are summarized, and the ratio of free Sphinganine to free sphingosine is discussed as a presumptive test for identifying animals consuming fumonisin-contami-nated feed.
-
fumonisin b1 inhibits sphingosine Sphinganine n acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ
Journal of Biological Chemistry, 1993Co-Authors: Alfred H. Merrill, Elaine Wang, G Van Echten, Konrad SandhoffAbstract:Abstract Fumonisins, mycotoxins produced by Fusarium moniliforme and a number of other fungi, cause neuronal degeneration, liver and renal toxicity, cancer, and other injury to animals. Recent work with rat hepatocytes (Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., and Merrill, A. H., Jr. (1991) J. Biol. Chem. 266, 14486-14490) found that fumonisins block sphingosine biosynthesis by inhibiting the conversion of Sphinganine to dihydroceramides, which precedes introduction of the 4,5-trans-double bond of sphingosine. The current study utilized mouse cerebellar neurons in culture to evaluate how this affects the distribution of newly synthesized ceramides among different complex sphingolipids. Fumonisin B1 inhibited ceramide synthase in mouse brain microsomes with a competitive-like kinetic behavior with respect to both Sphinganine and stearoyl-CoA. Fumonisin B1 inhibited sphingolipid biosynthesis in cultured cerebellar neurons in situ as reflected by accumulation of free Sphinganine, a reduction in the mass of total sphingolipids, reductions in the incorporation of [14C]serine into glucosylceramide, lactosylceramide, sphingomyelin, and gangliosides (GM1, GD3, GD1a, GD1b, GT1b, and GQ1b), and inhibition of the incorporation of [14C]galactose and [3H]Sphinganine into complex sphingolipids. Dose-response studies revealed that the labeling of sphingomyelin (IC50 of 0.7 microM) was more sensitive to inhibition by fumonisin B1 than was glycolipid formation (IC50 of approximately 7 microM) in these cells. A similar effect was seen when beta-fluoroalanine was added to inhibit the activity of serine palmitoyltransferase, the first enzyme of the pathway. The inhibition of complex sphingolipid synthesis was reversible, and nearly normal labeling profiles were obtained 48 h after removing the mycotoxin. These studies establish that fumonisin B1 inhibits de novo sphingolipid biosynthesis by neuronal cells and, moreover, that limiting ceramide synthesis differentially affects the formation of sphingomyelin versus glycosphingolipids.
E V Dyatlovitskaya - One of the best experts on this subject based on the ideXlab platform.
-
the sphingenine Sphinganine ratio in sphingolipids of transplantable rat tumors depends on a transplantation organ
Bioorganicheskaia khimiia, 2003Co-Authors: A G Kandyba, V A Koblyakov, E V DyatlovitskayaAbstract:The content of sphingenine (sphingosine) and Sphinganine was determined in the total pool of sphingomyelin and ceramide in the rat tumors transplanted subcutaneously and intrahepatically. The sphingenine/Sphinganine ratio in the subcutaneously transplanted sarcoma M1 and cholangiocellular carcinoma RS1 was lower than that in the sphingolipids of the intrahepatically transplanted tumors. However, the sphingenine/Sphinganine ratio in the subcutaneously transplanted rat hepatoma 27 was higher than in the intrahepatically transplanted hepatoma. These observations indicate that the sphingenine/Sphinganine ratio in sphingolipids of tumors depends on the tumor type and its cellular microenvironment.
-
The Sphingenine/Sphinganine Ratio in Sphingolipids of Transplantable Rat Tumors Depends on a Transplantation Organ
Bioorganicheskaia khimiia, 2003Co-Authors: A G Kandyba, V A Koblyakov, E V DyatlovitskayaAbstract:The content of sphingenine (sphingosine) and Sphinganine was determined in the total pool of sphingomyelin and ceramide in the rat tumors transplanted subcutaneously and intrahepatically. The sphingenine/Sphinganine ratio in the subcutaneously transplanted sarcoma M1 and cholangiocellular carcinoma RS1 was lower than that in the sphingolipids of the intrahepatically transplanted tumors. However, the sphingenine/Sphinganine ratio in the subcutaneously transplanted rat hepatoma 27 was higher than in the intrahepatically transplanted hepatoma. These observations indicate that the sphingenine/Sphinganine ratio in sphingolipids of tumors depends on the tumor type and its cellular microenvironment.
-
Sphinganine in sphingomyelins of tumors and mouse regenerating liver
Biochemistry, 2001Co-Authors: E V Dyatlovitskaya, A G Kandyba, A M Kozlov, O G SomovaAbstract:Contents of sphingenine (sphingosine) and Sphinganine were studied in sphingomyelins of transplantable mouse tumors (hepatoma-22, melanoma B16, Lewis lung carcinoma, intestine carcinoma) and rat nephroma RA. The content of Sphinganine was increased in sphingomyelins of hepatoma-22 and nephroma RA compared to sphingomyelins of liver and kidneys. Significant contents of Sphinganine were also found in sphingomyelins of other studied tumors. The content of Sphinganine in regenerating mouse liver (30 h after hepatectomy) was normal. The data suggest that disorders should exist in biosynthesis of sphingoid bases in tumors but not in normal rapidly proliferating tissue.
Konrad Sandhoff - One of the best experts on this subject based on the ideXlab platform.
-
synthesis of Sphinganine analogues modified in the head group
Tetrahedron, 1994Co-Authors: Thomas Kolter, Gerhild Van Echtendeckert, Konrad SandhoffAbstract:Abstract The synthesis of Sphinganine analogues modified in the head group is reported. The target compounds are efficiently prepared by means of the Henry reaction. Synthesis and results of preliminary investigations of the derivatives as potential inhibitors of sphingolipid biosynthesis are presented.
-
fumonisin b1 inhibits sphingosine Sphinganine n acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ
Journal of Biological Chemistry, 1993Co-Authors: Alfred H. Merrill, Elaine Wang, G Van Echten, Konrad SandhoffAbstract:Abstract Fumonisins, mycotoxins produced by Fusarium moniliforme and a number of other fungi, cause neuronal degeneration, liver and renal toxicity, cancer, and other injury to animals. Recent work with rat hepatocytes (Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., and Merrill, A. H., Jr. (1991) J. Biol. Chem. 266, 14486-14490) found that fumonisins block sphingosine biosynthesis by inhibiting the conversion of Sphinganine to dihydroceramides, which precedes introduction of the 4,5-trans-double bond of sphingosine. The current study utilized mouse cerebellar neurons in culture to evaluate how this affects the distribution of newly synthesized ceramides among different complex sphingolipids. Fumonisin B1 inhibited ceramide synthase in mouse brain microsomes with a competitive-like kinetic behavior with respect to both Sphinganine and stearoyl-CoA. Fumonisin B1 inhibited sphingolipid biosynthesis in cultured cerebellar neurons in situ as reflected by accumulation of free Sphinganine, a reduction in the mass of total sphingolipids, reductions in the incorporation of [14C]serine into glucosylceramide, lactosylceramide, sphingomyelin, and gangliosides (GM1, GD3, GD1a, GD1b, GT1b, and GQ1b), and inhibition of the incorporation of [14C]galactose and [3H]Sphinganine into complex sphingolipids. Dose-response studies revealed that the labeling of sphingomyelin (IC50 of 0.7 microM) was more sensitive to inhibition by fumonisin B1 than was glycolipid formation (IC50 of approximately 7 microM) in these cells. A similar effect was seen when beta-fluoroalanine was added to inhibit the activity of serine palmitoyltransferase, the first enzyme of the pathway. The inhibition of complex sphingolipid synthesis was reversible, and nearly normal labeling profiles were obtained 48 h after removing the mycotoxin. These studies establish that fumonisin B1 inhibits de novo sphingolipid biosynthesis by neuronal cells and, moreover, that limiting ceramide synthesis differentially affects the formation of sphingomyelin versus glycosphingolipids.
-
biosynthesis of sphingolipids dihydroceramide and not Sphinganine is desaturated by cultured cells
Biochemical and Biophysical Research Communications, 1992Co-Authors: Jiirgen Rother, Gerhild Van Echten, Giinter Schwarzmann, Konrad SandhoffAbstract:Radioactively labeled N-[1-14C]-octanoyll-Sphinganine and D-erythro-[3-3H]-Sphinganine were administered in parallel experiments to neuroblastoma cells B 104. A time dependent formation of ceramide with a double bond in its sphingoid backbone was observed in both cases. In the presence of fumonisin B1 (25 μM), a strong inhibitor of Sphinganine N-acyltransferase, desaturated ceramide was formed only when cells were fed with N-[1-14C]-octanoyl-Sphinganine but not with [3-3H]-Sphinganine. Thus, the introduction of the double bond occurs only at the level of dihydroceramide, after N-acylation of Sphinganine. It is now obvious that sphingosine is not a biosynthetic intermediate but exclusively a catabolic product of cellular sphingolipids.
-
subcellular localization and membrane topology of serine palmitoyltransferase 3 dehydroSphinganine reductase and Sphinganine n acyltransferase in mouse liver
Journal of Biological Chemistry, 1992Co-Authors: E C Mandon, I Ehses, J Rother, G Van Echten, Konrad SandhoffAbstract:Abstract Serine palmitoyltransferase, 3-dehydroSphinganine reductase and Sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxoSphinganine, Sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydroSphinganine reductase, and Sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydroSphinganine reductase, and Sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and Sphinganine N-acyltransferase activities by 30-70%.
Guy P. Mannaerts - One of the best experts on this subject based on the ideXlab platform.
-
Do sphingoid bases interact with the peroxisome proliferator activated receptor α (PPAR-α)?
Cellular Signalling, 2000Co-Authors: Paul P Van Veldhoven, Guy P. Mannaerts, Peter Declercq, Myriam BaesAbstract:Abstract In a search for possible endogenous ligands of nuclear receptors that are activated by peroxisome proliferators (PPARs), a solid phase binding assay was developed employing recombinant mouse PPAR-α, containing a myc-epitope, a histidine repeat and a kinase A domain. After in vitro labelling with 32P-γ-ATP, the binding of purified 32P-PPAR-α to a panel of different natural and synthetic lipids, immobilized on silica layers, was evaluated. Autoradiographs of the silica layers revealed binding to two main classes of lipophilic compounds. A first class comprised (poly)unsaturated fatty acids. Compounds belonging to a second class were characterized by the presence of an overall positive charge such as long chain amines, sphingoid bases (sphingenine), and lysoglycosphingolipids (psychosine). PPAR-α did not bind to N-acylated sphingoid bases (ceramides) or to sphingenine phosphorylated at the primary hydroxy group (sphingenine-1-phosphate). The binding of PPAR-α to sphingoid bases might be of interest given the role of PPAR-α and sphingolipids in various cellular processes.
-
identification and subcellular localization of Sphinganine phosphatases in rat liver
Biochemical Journal, 1995Co-Authors: P De Ceuster, Guy P. Mannaerts, Paul P Van VeldhovenAbstract:One of the primary products of [4,5-3H]Sphinganine phosphate, added to fibroblast cultures, is Sphinganine [Van Veldhoven and Mannaerts (1994) Biochem. J. 299, 597-601], implicating the physiological action of (a) hitherto unknown phosphatase(s). We have now further characterized this activity in rat liver. In homogenates, the dephosphorylation appeared to be catalysed by multiple enzymes. A low-affinity system was active at acidic pH, whereas at physiological pH values hydrolysis was carried out by a high-affinity enzyme. The latter was sensitive to Zn2+ and detergents and possessed a pH optimum of 7.5. Upon cell fractionation the major portion of the high-affinity activity was recovered in the nuclear and microsomal fractions. Further separation of the microsomal fraction showed an association predominantly with vesicles derived from the plasma membrane. Likewise, when plasma membranes were prepared from the nuclear fraction, the high-affinity phosphatase co-purified with the plasma membrane markers. From the differential effects of bivalent cations, chelators, water-soluble and amphiphilic phosphate esters, detergents and other compounds, it could be concluded that the plasma membrane-associated Sphinganine-phosphatase activity is not due to alkaline phosphatase, dolichol-phosphatase, the N-ethylmaleimide-insensitive phosphatidate phosphatase or ceramide-phosphatase. The dephosphorylation observed at acidic pH in homogenates appeared also to be enriched in purified plasma membranes and might represent a side-activity of ceramide-phosphatase. We speculate that the high-affinity phosphatase, which is especially active in neuronal tissues, plays a role in the attenuation of bioactive phosphorylated sphingoid bases such as sphingenine phosphate, and propose to name it sphingosine-phosphatase.
-
on the presence of phosphorylated sphingoid bases in rat tissues a mass spectrometric approach
FEBS Letters, 1994Co-Authors: Paul P Van Veldhoven, Guy P. Mannaerts, Patrick De Ceuster, Raoul Rozenberg, Edmond De HoffmannAbstract:A simple and straightforward procedure to analyze phosphorylated sphingoid bases has been developed. After phase separation of lipid extracts under alkaline conditions, the compounds were quantitatively recovered in the aqueous upper phase. Following a clean-up of the aqueous phase on C18-solid phase extraction columns, the amino-group of the bases was derivatized by means of phenylisothiocyanate addition. FAB-MS of the phenylthiocarbamate derivatives of sphingenine- and Sphinganine-phosphate in the negative mode revealed the expected pseudo-molecular ions (M-1) at 513 m/z and 515 m/z, respectively. Moreover, a typical fragmentation pattern, characterized by the loss of the phenylthiocarbamate moiety (m/z = 135), was observed. When applied to rat tissues, the presence of sphingenine-phosphate in brain, kidney and liver could easily be demonstrated. Highest levels, amounting to 5 nmol/g of wet weight, were present in brain.
-
Sphinganine 1 phosphate metabolism in cultured skin fibroblasts evidence for the existence of a sphingosine phosphatase
Biochemical Journal, 1994Co-Authors: Paul P Van Veldhoven, Guy P. MannaertsAbstract:On addition of [4,5-3H]Sphinganine 1-phosphate to human fibroblast monolayers, the label was efficiently removed from the culture medium. In contrast with the reported stability of phosphorylated sphingenine in 3T3 cells [Desai, Zhang, Olivera, Mattie and Spiegel (1992). J. Biol. Chem. 267, 23122-23128] and B16 melanoma cells [Sadahira, Ruan, Hakomuri and Igarashi (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9686-9690], Sphinganine 1-phosphate appeared to be subjected to a fast and extensive metabolism in fibroblasts, the major pathways being cleavage and dephosphorylation. The first of these pathways, catalysed by sphingosine-phosphate lyase, resulted in the formation of labelled palmitaldehyde, which was recovered, mainly after oxidation, in glycerophospholipids in an ester bond. A smaller part of the palmitaldehyde was reduced and incorporated in alk(en)ylphospholipids. Dephosphorylation of spinganine 1-phosphate, a hitherto overlooked pathway catalysed by an unknown phosphatase(s), gave rise to Sphinganine, which was converted by N-acylation into ceramide and then incorporated in spingomyelin and glycosphingolipids.
