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F Schuber

Publications and source records attributed to F Schuber.

At least 55 records · Page 3Linked to original sources
PubMed1992

Preferential cyclization of 2,3(S):22(S),23-dioxidosqualene by mammalian 2,3-oxidosqualene-lanosterol cyclase.

Kinetic studies on the cyclization of 2,3(S)-oxido and 2,3(S):22(S),23-dioxido[14C]squalene catalyzed by liver oxidosqualene-lanosterol cyclase revealed a specificity (in terms of V/Km) of the enzyme for the diepoxide. The specificity ratio was dependent on the enzyme preparation, i.e. purified or microsomal, and was highest (about 5) with the microsomal enzyme in the presence of supernatant protein factors. These results explain why, in the presence of cyclase inhibitors, the squalene epoxides can be channeled into a cholesterol biosynthesis regulatory pathway via 24(S),25-epoxylanosterol and 24(S),25-epoxycholesterol.

3T3 Cells↗
PubMed1992

Enantioselectivity of the hydrolysis of linoleic acid monoepoxides catalyzed by soybean fatty acid epoxide hydrolase.

Soybean epoxide hydrolase efficiently catalyzes the hydration of the two positional isomers of linoleic acid monoepoxides into their corresponding vic-diols. Kinetic analysis of the progress curves, obtained at low substrate concentrations (i.e. [So] much less than Km), and analysis of the residual substrates by chiral-phase HPLC, indicate that the hydrolase is highly enantioselective, i.e. cis-9R,10S-epoxy-12(Z)-octadecenoic and cis-12R,13S-epoxy-9(Z)-octadecenoic acids are preferentially hydrolyzed (the enantioselectivity ratios are 15 and 28, respectively). Importantly, these two enantiomers are the one formed preponderantly by epoxidation of linoleic acid by peroxygenase, a hydroperoxide-dependent oxidase we have previously described in soybean (Blée, E., and Schuber, F., Biochem. Biophys. Res. Commun. (1990) 173, 1354-1360).

Chromatography, High Pressure Liquid↗
PubMed1992

Regio- and enantioselectivity of soybean fatty acid epoxide hydrolase.

Soluble epoxide hydrolase purified from soybean catalyzes trans-addition of water across the oxirane ring of cis-9,10-epoxystearic acid with inversion of configuration at the attacked carbon, yielding threo-9,10-dihydroxystearic acid. Kinetic analyses of the progress curves, obtained at low substrate concentrations (i.e. [S] much less than Km), and determination of the enantiomeric excess of the residual substrate by chiral-phase high-performance liquid chromatography at different reaction times, indicate that the epoxide hydrolase hydrates preferentially cis-9R, 10S-epoxystearic acid (V/Km ratio, approximately 20). Interestingly, this enantiomer is obtained by epoxidation of oleic acid catalyzed by peroxygenase, a hydroperoxide-dependent oxidase, we have previously described in soybean (Blée, E., and Schuber, F. (1990) J.Biol. Chem. 265, 12887-12894). For the epoxide hydrolase to show high enantioselectivity there must be a free carboxylic acid functionality on the substrate which probably influences its positioning within the active site. This selectivity, which in principle can be used for kinetic resolution of the cis-9,10-epoxystearic acid enantiomers, is much reduced with methyl cis-9,10-epoxystearate. 18O-Labeling experiments indicate that water attacks both cis-9,10-epoxystearic acid enantiomers on the oxirane carbon which has the S-chirality. Results show that soybean epoxide hydrolase produces exclusively threo-9R,10R-dihydroxystearic acid, i.e. a naturally occurring metabolite in higher plants. cis-9,10-Epoxy-18-hydroxystearic acid, a cutin monomer, was a poorer substrate of the epoxide hydrolase than 9,10-epoxystearic acid (V/Km ratio for the preferred enantiomers, approximately 19). From a physiological point of view, peroxygenase and this newly described epoxide hydrolase could be responsible, in vivo, for the biosynthesis of a class of oxygenated fatty acid compounds known to be involved in cutin monomers production and in plant defense mechanisms.

Chromatography, High Pressure Liquid↗
PubMed1992

Slow-binding inhibition of NAD+ glycohydrolase by arabino analogues of beta-NAD.

Modifications at the 2'-position of the nicotinamide-ribosyl moiety influence dramatically the nature of the interactions of the modified beta-NAD+ with calf spleen NAD+ glycohydrolase (EC 3.2.2.6), an enzyme that cleaves the nicotinamide-ribose bound in NAD(P)+. Nicotinamide arabinoside adenine dinucleotide (ara-NAD+) and nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide (araF-NAD+) are not hydrolyzed at measurable rates and are the first documented examples of reversible slow binding inhibitors of this class of enzyme. The kinetic data obtained are consistent with both slow kon and koff rate constants in the formation of an enzyme-inhibitor complex, i.e. the association rate constants are about 10(4) and 10(6) slower than diffusion rates, respectively, for araF-NAD+ and ara-NAD+, and the half-life of the complex is about 3-10 min for both analogues. The kinetic model does not account for a slow turnover of an ADP-ribosyl-enzyme intermediary complex. AraF-NAD+ is one of the most potent inhibitors described for NAD+ glycohydrolase.

Animals↗
PubMed1992

Occurrence of fatty acid epoxide hydrolases in soybean (Glycine max). Purification and characterization of the soluble form.

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.

Epoxide Hydrolases↗
PubMed1991

Parameters affecting the immunogenicity of a liposome-associated synthetic hexapeptide antigen.

The effect of liposome association on the immunogenicity of the hexapeptide IRGERA was investigated. When administered in the absence of a carrier and adjuvant this peptide, which corresponds to a linear epitope located at the C-terminus of histone H3, was not immunogenic. When mice were immunized with the peptide covalently linked to the surface of small unilamellar vesicles containing monophosphoryl lipid A as adjuvant, a relatively long-lasting response with memory cell induction was observed. The anti-peptide antibodies raised in this way reacted with the cognate sequence in the native histone. In contrast, coupling of the peptide to the surface of large vesicles yielded both an IgM and IgG response of short duration whereas encapsulation of the free peptide in large vesicles was ineffective. These results indicate that with short synthetic peptides, liposomes provide a substitute for a carrier protein. However, an adjuvant has to be incorporated in the vesicles in order to obtain an efficient immune response. Such an approach may be useful for designing synthetic vaccines.

Adjuvants, Immunologic↗
PubMed1991

Influence of the galactosyl ligand structure on the interaction of galactosylated liposomes with mouse peritoneal macrophages.

Liposomes bearing at their surface mono- and triantennary galactosyl ligands were prepared and their interaction with the galactose receptor of mouse peritoneal macrophages studied. Triantennary structures were synthesized by coupling derivatives of 1-thio-beta-D-galactose to the amino groups of lysyl-lysine dipeptide. Galactosylated liposomes were obtained either by synthesis of neo-galactolipids followed by their incorporation into the vesicles or by neo-galactosylation of performed liposomes by reaction between thiol-functionalized galactosyl ligands and vesicles bearing maleimido groups. The interaction of the galactosylated liposomes with the macrophage lectin was remarkably sensitive to the topology of the ligands, i.e., a spacer-arm length about 3 nm was necessary and, in contrast to results obtained with the galactose receptor of other cells, the triantennary structure did not provide additional binding. Related to the strategy of drug delivery with targeted liposomes, these results indicate that lectins from different cells might possibly be distinguished by using multiantennary ligands having optimal geometries.

Animals↗
PubMed1990

Stereochemistry of the epoxidation of fatty acids catalyzed by soybean peroxygenase.

The stereochemistry of C18 unsaturated fatty acids epoxidation catalyzed by detergent-solubilized and partially purified soybean peroxygenase was determined by chiral phase HPLC. Linoleic acid was oxidized into 9, 10- and 12,13-cis-epoxyoctadecenoic acids with a high enantiofacial selectivity. A 5.2:1 and 2.3:1 ratio respectively in favor of the 9(R), 10(S)- and 12(R), 13(S)-epoxy enantiomers was observed. These epoxy-derivatives of linoleic acid have the chirality of metabolites known to be involved in plant defense against fungi. This finding is of importance in establishing a physiological role for the peroxygenase.

Chromatography, High Pressure Liquid↗
PubMed1990

Efficient epoxidation of unsaturated fatty acids by a hydroperoxide-dependent oxygenase.

Detergent-solubilized and partially purified soybean peroxygenase was shown to actively catalyze, in the presence of alkylhydroperoxides as co-substrates, the epoxidation of mono- and polyunsaturated fatty acids such as oleic and linoleic acids. Octadecenoic acids were found to be better substrates than shorter mono-unsaturated fatty acids (C16:1 or C14:1), but the position of their double bond (at positions 6, 9, or 11) had little effect on the rates of epoxidation. The peroxygenase exhibits a strong stereospecificity since octadecenoic acids with double bonds in trans-configuration were not epoxidized at detectable rates. Oxidation of linoleic acid yielded the two positional monoepoxide isomers and, as the minor product, the diepoxide. An important regioselectivity was, however, observed in this case; i.e. the unsaturation at position 9,10 was epoxidized preferentially to that at 12, 13. Oxidation of oleic acid in the presence of 18O-labeled hydroperoxy-linoleic acid revealed an incorporation of about 80% of the label into the epoxide ring. Products similar to those formed by the peroxygenase by epoxidation of unsaturated free fatty acids such as linoleic acid have been described as important metabolites (leukotoxins) in the defense of plants, e.g. in fungal agressions. This aspect underlines the physiological relevance of this new and potent catalytic activity of the peroxygenase.

Epoxy Compounds↗
PubMed1990

Fate of ecto-NAD+ glycohydrolase during phagocytosis of normal and mannosylated latex beads by murine macrophages.

In order to gain a better understanding of the role of ecto-NAD+ glycohydrolase, an enzyme predominantly associated with phagocytic cells, we have studied its fate in murine macrophages (splenic, resident peritoneal and Kupffer cells) during phagocytosis of opsonized on mannosylated latex beads. In parallel, we have also monitored nucleotide pyrophosphatase, another ecto-enzyme of macrophages. Phagosomes were isolated by flotation in a discontinuous sucrose gradient and the enzyme activities were determined with fluorometric methods. Low levels of NAD+ glycohydrolase and nucleotide pyrophosphatase could be measured associated with the phagosomal fractions, eg, respectively less than 4.5% and 10% in spleen macrophages. The phagosomal activities originate from the plasma membrane, ie they were latent and inactivation of ecto-NAD+ glycohydrolase with the diazonium salt of sulfanilic acid resulted in a marked decrease of this enzyme activity in the phagosomal fractions. Pre-labelling of the cell surface by [3H]-galactosylation indicated that NAD+ glycohydrolase is internalized to a lesser extent than an average surface-membrane unit. These results indicate that if ecto-NAD+ glycohydrolase of macrophages can be internalized to a limited extent during phagocytosis of opsonized or mannosylated latex beads, this enzyme appears to be predominantly excluded from the surface area involved in the uptake of such particles.

Animals↗
PubMed1989

Neo-mannosylated liposomes: synthesis and interaction with mouse Kupffer cells and resident peritoneal macrophages.

In order to target liposomes to cells expressing at their surface mannose receptors, e.g. mouse Kupffer cells and peritoneal macrophages, we have developed a new synthetic strategy which allows a chemically well defined preparation of neo-mannosylated vesicles. alpha-D-Thiomannopyranoside residues, substituted with a hydrophilic spacer arm and functionalized with a sulfhydryl group, were covalently coupled to preformed large unilamellar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. Liposomes, containing 15 mol% of mannosyl residues, were specifically aggregated with concanavalin A; this aggregation could be reversed by an excess of free methyl alpha-D-mannopyranoside indicating that the surface ligands were freely accessible to the lectin. The neo-mannosylated liposomes presented in vitro an increased binding to cells possessing alpha-D-mannose specific binding sites. At 37 degrees C a specific binding, up to 9-fold compared to control vesicles, was observed. These neo-mannosylated vesicles represent attractive tools for targeting bio-active molecules to macrophage-associated diseases.

Animals↗
PubMed1989

Squalene epoxide cyclase and microemulsion.

Squalene epoxide cyclase was extracted from microsomal preparations of rat liver using anionic, cationic and non-ionic microemulsions. The anionic microemulsion was the best with respect to protein solubilisation, extracted cyclase activity and stability of this activity over time. The activity assay showed cyclase activity to be higher in anionic microemulsion than in buffer in the presence of surfactant. Calcium chloride in the anionic microemulsion had a stabilising effect and less total protein seemed to be extracted.

Animals↗
PubMed1988

Inhibition by the fungicide fenpropimorph of cholesterol biosynthesis in 3T3 fibroblasts.

Fenpropimorph (N-[3-(p-t-butylphenyl)-2-methylpropyl]-cis-2,6-dimethylmorpholine), a morpholine fungicide known to be an inhibitor of sterol biosynthesis in fungi and in higher plants, was demonstrated to be an efficient inhibitor of cholesterol biosynthesis in cultured Swiss 3T3 fibroblasts. Treatment of the mammalian cells with fenpropimorph resulted in a dose-dependent inhibition of [14C]acetate incorporation into the C27 sterols [IC50 (concentration causing half-maximal inhibition) = 0.5 microM], which was accompanied by an accumulation of polar sterols and a decrease in cellular hydroxymethylglutaryl-CoA reductase activity. Exposure of the cells to the drug affected cell growth. Analysis of the sterols in the growth-arrested and in the pulse-labelled cells indicate that fenpropimorph has, in the sterol-biosynthetic pathway, target enzymes in mammalian cells different from those in the other phyla. Whereas in plants and fungi fenpropimorph mainly affects sterol isomerases and reductases, in the fibroblasts its main target seems to be the demethylation of lanosterol.

Acetates↗
PubMed1988

Preparation of neo-galactosylated liposomes and their interaction with mouse peritoneal macrophages.

In order to target liposomes to cells expressing at their surface a galactose-binding site we have prepared liposomes containing new synthetic galactolipids. Neo-galactosylated liposomes were prepared by covalently coupling beta-D-1-thiogalactopyranoside residues, substituted with a hydrophilic spacer-arm and functionalized with a sulfhydryl group, to preformed large unilamelar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. The vesicles, having a galactose content above a threshold value of about 5 mol%, could be aggregated with Ricinus communis agglutinin. This aggregation was reversed by addition of excess free methyl beta-D-galactopyranoside, indicating that the surface glucidic moieties of these liposomes were accessible to the lectin. Compared to the control vesicles, the neo-galactosylated liposomes (containing 15 mol% galactose) presented in vitro an increased binding to cell possessing a beta-D-galactose specific receptor, i.e. resident mouse peritoneal macrophages. At 4 degrees C, the specific binding was about 2-fold, whereas at 37 degrees C it was increased to about 4-5-fold. This differential binding was largely unaffected by serum and, interestingly was much dependent on the degree of galactosylation of the liposomes, i.e. a threshold value of 5 mol% was needed to observe an increased binding of the targeted vesicles to the macrophages.

Animals↗
PubMed1988

In vitro drug delivery mediated by ecto-NAD+-glycohydrolase ligand-targeted liposomes.

We have studied the growth-inhibitory potency of methotrexate and methotrexate-gamma-aspartate encapsulated in liposomes conjugated to ligands of ecto-NAD+-glycohydrolase (Salord, J. et al., Biochim. Biophys. Acta 886 (1986) 64-75). The ability of targeted liposomes to enhance growth inhibition, which amounted to a 4-fold reduction of the drug concentration required to inhibit cell growth by 50% as compared to nontargeted liposomes, was observed only with cells expressing this ecto-enzyme activity, i.e., Swiss 3T3 fibroblasts and RAJI, a Burkitt-type lymphoma cell line. Delivery of the encapsulated drugs was inhibited by NH4Cl and varied with the endocytic capacity of the cells. Only small unilamellar vesicles affected the growth of the lymphoma cells, whereas the fibroblasts were more sensitive to large unilamellar vesicles. With vesicles of appropriate size, there was a good correlation between the specific binding of the targeted liposomes to cells and drug delivery. Our results suggest that ecto-NAD+-glycohydrolase can provide a recognition site on target cells and mediate the internalization of targeted liposomes by a mechanism most probably related to adsorptive endocytosis.

Ammonium Chloride↗
PubMed1988

Potent inhibition of cholesterol biosynthesis in 3T3 fibroblasts by N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol, a new 2,3-oxidosqualene cyclase inhibitor.

N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol, a new compound rationally designed to inhibit the 2,3-oxidosqualene cyclase (M. Taton et al., Biochem. biophys. Res. Commun. 138, 764, 1986) was studied as an inhibitor of cholesterol biosynthesis in Swiss 3T3 fibroblasts. Treatment of cells, which were grown for 2 days in a delipidated medium, resulted in a dramatic decrease of [14C]acetate incorporation into the C27-sterol fraction. An IC50 of 20 nM was calculated, which classes this drug amongst the most powerful cholesterol biosynthesis inhibitors acting at the 2,3-oxidosqualene-lanosterol cyclase tested so far on mammalian cells. The inhibition of the C27-sterols synthesis was correlated with the accumulation of 2,3-[14C]oxidosqualene and of 2,3:22,23-[14C]dioxidosqualene indicating that the cyclase was indeed an intracellular target of the drug. A minor secondary target was identified as the sterol-8-ene isomerase. Cells treated with the inhibitor also accumulated sterols more polar than cholesterol which could originate, for example, from the metabolization of 2,3:22,23-dioxidosqualene. Treatment of the cells with increasing concentrations of the drug resulted in a progressive reduction of the HMG-CoA reductase activity (up to 50% of control). The drug affected normal growth of the fibroblasts and growth arrest was correlated with a decrease in cellular cholesterol content to less than 50% of control. This work indicates that N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol is a potent and promising new tool in the inhibition of cholesterol biosynthesis in mammalian cells.

Anticholesteremic Agents↗