Metabolism of alkyl glyceryl ethers and their noninvolvement in alkane biosynthesis in plants

[1-¹⁴C]Octadecyl glyceryl ether did not label alkanes in the leaves of Brassica oleracea and Pisum sativum while [1-¹⁴C]octadecanol and [1-¹⁴C]octadecanoic acid readily labeled the alkanes. About 40% of the exogenous-labeled glyceryl ether was incorporated intact into choline phosphatide while 10–20% was converted into fatty acids and alcohols. [1-¹⁴C]octadecanol was not converted into alkyl glyceryl ether, but it was oxidized to the corresponding acid and then incorporated into alkanes. These results show that alkyl ether is not an intermediate in alkane biosynthesis. When [1-¹⁴C-1-³H]-octadecanol was fed to the leaves of B. oleracea and P. sativum, only the ¹⁴C and no ³H was incorporated into alkanes, ketones, and secondary alcohols. These results show that fatty alcohols are first oxidized to the acid before being incorporated into alkanes, ruling out fatty alcohol, alkyl ether, and alk-1-enyl ether as intermediates in alkane biosynthesis. The exogenous alcohols were also readily esterified into wax esters in both tissues.     

Metabolism of the alkane analogue n-dioctyl ether by Acinetobacter species.

Metabolism of n-dioctyl ether by Acinetobacter species HO1-N resulted in formation of 8-n-octoxy-1-octanoic acid and 2-n-octoxy-1-acetic acid. The 16-carbon ether acid was incorporated into the cellular lipids, whereas the 10-carbon ether acid accumulated in the growth medium. Qualitative and quantitative characteristics of the cellular phospholipids were similar to hexadecane-grown cells. The growth of Acinetobacter on dioctyl ether occurred at the expense of six-carbon atoms of dioctyl ether.     

Effect of anti-tumor ether lipids on ordered domains in model membranes

1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (OMPC, edelfosine) and 1-hexadecylphosphocholine (HePC, miltefosine) represent two groups of synthetic ether lipid analogues with anti-tumor activity. Because of their hydrophobic nature, they may become incorporated into plasma membranes of cells, and it has been argued that they may act via association with lipid rafts. With the quenching of steady-state fluorescence of probes preferentially partitioning into sterol-rich ordered domains (cholestatrienol and trans-parinaric acid), we showed that OMPC and HePC by themselves did not form sterol-rich domains in fluid model membranes, in contrast to the two chain ether lipid 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine. Nevertheless, all three ether lipids significantly stabilized palmitoyl-sphingomyelin/cholesterol-rich domains against temperature induced melting. In conclusion, this study shows that anti-tumor ether lipids are likely to affect the properties of cholesterol-sphingomyelin domains (i.e., lipid rafts) when incorporated into cell membranes.     

Polyaza crown ether as non-nucleosidic building blocks in DNA-conjugates

The synthesis of amphiphilic polyaza crown ether monomers X (palmityl-substituted), Y (cholesteryl-substituted) and Z (dipalmityl-subtituted) and their incorporation into oligonucleotides are described. Their effects on thermal duplex stability were investigated by UV melting curve analysis. Thermal denaturation experiments showed remarkable stabilization of dsDNA by polyaza crown ether monomers when incorporated in opposite positions. The series of polyaza crown ether monomers (X, Y, and Z) with different lipophilicity showed a trend of increased stability of the corresponding dsDNA with increasing lipophilicity of the polyaza crown ether monomer.     

Effect of phorbolmyristate acetate on fatty acid uptake and distribution in human promyelocytic leukemia (HL-60) cells in vitro

Human promyelocytic leukemia (HL-60) cells can be induced to differentiate to macrophages in vitro by phorbolmyristate acetate (PMA). HL-60 cells, unlike normal cells incorporated a major portion of linoleic acid (LA) and arachidonic acid (AA) in the ether lipid fraction. On exposure to PMA, similar to the normal cells tested, the fatty acids were incorporated mainly in the phospholipid fraction. Since, ether lipid pool is metabolically inert and considered as a storage pool where as the phospholipid fraction is a metabolically active pool this may explain, at least in part, the low metabolic rate of AA and the low phospholipase A2 activity in HL-60 cells.     

Synthesis of an asymmetrically substituted AZA crown ether as metal and amino acid binding site in DNA conjugates

Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.     

Clathrin-Dependent and Clathrin-Independent Endocytosis are Differentially Sensitive to Insertion of Poly (Ethylene Glycol)-Derivatized Cholesterol in the Plasma Membrane

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036–3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.     

Metabolism of glycerol ether-containing lipids in dog fish (Squalus acanthias)

Dogfish (Squalus acanthias) received intrahepatic injections of either palmitic acid-1-(14)C or chimyl alcohol-1-(14)C. The lipids of the liver were then analyzed for incorporated radioactivity. The experiments with labeled palmitic acid demonstrated that fatty acids are reductively incorporated into the alkyl and alkenyl ether chains of glycerolipids. Significantly lower specific activities were found for the diacyl alk-1'-enyl ethers and diacyl glycerol ethers than for other glycerol ether-containing lipids. These compounds may therefore represent terminal points in ether-lipid metabolism. The studies with labeled chimyl alcohol indicate that dogfish liver contains enzymes that have a high capacity for oxidatively cleaving alkyl ether linkages. Furthermore, it is probable that alkyl ethers are converted directly to alkenyl ethers, possibly via a biodehydrogenation reaction.     

Metabolism of orally administered rac-1-O-[1'-14C]dodecylglycerol and nutritional effects of dietary rac-1-O-dodecylglycerol in mice

The metabolism of orally administered rac-1-O-[1'-14C]dodecylglycerol was investigated in mice. The substrate was rapidly absorbed in the intestine and then incorporated into ether glycerolipids of various organs and tissues in high proportions. In intestine and liver, however, large amounts of rac-1-O-[1'-14C]dodecylglycerol were catabolized by oxidative cleavage of the ether bond followed by degradation of the radioactive cleavage product, i.e., lauric acid, to water-soluble metabolites that were excreted in the urine at a fast rate. The feeding of a rac-1-O-dodecylglycerol-containing diet (1 g rac-1-O-dodecylglycerol/kg body weight X day) given over a period of 4 weeks did not significantly alter body weights or organ weights of mice. Analysis of total lipids revealed that high proportions of the substrate were incorporated into ether lipids of all organs and tissues during the feeding period, generally accompanied by a remarkable increase in saturated acyl moieties and a concomitant decrease of linoleoyl moieties of total lipids. Yet, 4 weeks after removal of the rac-1-O-dodecylglycerol-containing diet, the lipids of murine organs and tissues showed a close resemblance to those of the control group.     

Prenylated proteins from kidney

When [5-3H]mevalonate is injected into mice, it is incorporated into macromolecules in the kidney. The incorporated material is stable to treatment with RNase or DNase but not protease, indicating that the radioactivity is associated with protein. Electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels indicates a molecular weight of about 25,000. The incorporated radioactivity can be released from the polypeptide and extracted into organic solvents after hydrolysis with acid or base or by treatment with protease. The conditions required for hydrolysis strongly suggest that the linkage between the protein and the mevalonate-derived material is an allylic ether. The chromatographic mobility of the incorporated material in several systems is similar to that of dolichol C95.     

Toward a catalytic site in DNA: polyaza crown ether as non-nucleosidic building blocks in DNA conjugates

A number of functionalized polyaza crown ether building blocks have been incorporated into DNA-conjugates as catalytic Cu(2+) binding sites. The effect of the DNA-conjugate catalyst on the stereochemical outcome of a Cu(2+)-catalyzed Diels-Alder reaction will be presented.     

Synthesis and biological properties of the fluorescent ether lipid precursor 1-O-[9'-(1'-pyrenyl)]nonyl-sn-glycerol

The synthesis of an omega-pyrene-labeled 1-O-alkyl-sn-glycerol was performed using a chirospecific method starting from R-(-)-2,3-O-isopropylidene-sn-glycerol. The product, 1-O-[9'-(1'-pyrenyl)]nonyl-sn-glycerol (pAG), is a fluorescent ether lipid that has a pyrene moiety covalently attached at the alkyl chain terminus. pAG was taken into CHO-K1 cells and a plasmalogen-deficient variant of CHO-K1, NRel-4. This variant is defective in dihydroxyacetonephosphate acyltransferase, which catalyzes the first step in plasmenylethanolamine (PlsEtn) biosynthesis. pAG was incorporated primarily into ethanolamine and choline phospholipids as well as a neutral lipid fraction tentatively identified as alkyldiacylglycerol. NRel-4 accumulated more fluorescence in the phospholipid fraction than CHO-K1, specifically in the ethanolamine phospholipids. Analysis of the fluorescent lipids showed that 93% of the pAG was incorporated into glycerolipids with the ether bond intact. Although the addition of 20 microM 1-O-hexadecyl-sn-glycerol to the medium fully restored PlsEtn biosynthesis in NRel-4 cells, pAG only partially restored PlsEtn synthesis. Incubation of cells with pAG followed by irradiation with long-wavelength (>300 nm) ultraviolet light resulted in cytotoxicity. NRel-4 cells displayed an increased sensitivity to this treatment compared with CHO-K1 cells. This photodynamic cytotoxicity approach could be used to select for mutants that are defective in downstream steps in ether lipid biosynthesis.     

Synthesis of an Asymmetrically Substituted AZA Crown Ether as Metal and Amino Acid Binding Site in DNA Conjugates

Abstract

Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.     

Untersuchungen über den Stickstoffstoffwechsel von Pansenprotozoen (Entodinium) mittels 14C-Karbonatinkorporation

SYNOPSIS. Isolated rumen protozoan species (Entodinium) were incubated in vitro with starch, antibiotics and Na₂¹⁴CO₃. ¹⁴C-carbonate was incorporated into amino acids of the Entodinium protein. Arginine, aspartic acid, alanine, and glutamic acid had the highest specific activities. Carbonate was incorporated into the TCA-soluble fraction, into cell polysaccharides, and into ether and acetone extracts of TCA-precipitates.     

Asymmetric disposition of detergents within vesicle bilayer and its effect on ion permeation through the membrane

Phospholipid vesicles were prepared by detergent removal using hydrophobic porous beads, Amberlite XAD-2, or dialysis from detergent-phospholipid mixed micelles. The liposomes formed were found to be mostly unilammellar vesicles. The vesicle diameter was estimated, by both quasi-elastic light-scattering and gel-exclusion chromatography on Sephacryl S-1000, to be 80 nm for the vesicles formed by removal of octaethylene glycol monododecyl ether by the bead method. The effect of detergents within a bilayer on ion permeation was demonstrated. When the content of octaethylene glycol monododecyl ether reached a molar ratio of 0.2, the intrinsic ion selectivity of the phospholipid membrane between anion and cation was diminished. The ion permeability measured for vesicles with detergent incorporated into initially detergent-free vesicles was about 10-times greater than that for vesicles with detergent remaining following the process of detergent removal. This observation was explained by the different disposition of the detergent in the bilayer, that is, when vesicles were formed by the removal of detergent from mixed micelles, the residual detergent became distributed in both the outer and inner leaflets, and when the detergent was incorporated into initially detergent-free vesicles, the detergent became distributed only in the outer leaflet within the experimental time limits. This idea was supported by the NMR studies. It was also found that, as a detergent, octaethylene glycol monododecyl ether has a stronger effect on ion permeation than octyl glucoside.     

Synthesis of the spin-labeled derivative of an ether-linked phospholipid possessing high antineoplastic activity.

We report here the complete synthesis of the spin-labeled derivative of an antitumor ether phospholipid, 1-O-octadecyl-2-O-(4'-doxylpentyl)-rac-glycerol-3-phosphocholine. This also represents the first time that the synthesis of a nitroxide spin-labeled diether phospholipid is described. In vitro experiments showed that at micromolar concentrations, this new analog is readily incorporated into the plasma membranes of human HL60 and mouse E8/AK.D1 leukemic cells, and subsequently kills the cells. The availability of this new probe should permit the electron spin resonance spectroscopic approach to investigate ways by which anti-tumor ether phospholipids selectively destroy the tumor cells.     

Biosynthesis and utilization of long-chain alcohols in rat brain: aspects of chain length specificity

Chain Length specificity in alkyl glycerolipid biosynthesis was studied with microsomal preparations from 19-day-old rat brain. Saturated alcohols ranging from 12 to 22 carbon atoms were incorporated at different rates into alkyl dihydroxyacetone phosphate, the first intermediate in ether lipid biosynthesis. The rate of alkyl dihydroxyacetone phosphate formation was highest with hexadecanol and alcohols of either longer or shorter chain length were utilized much less efficiently. The monounsaturated octadecenol was incorporated more readily than any of the saturated alcohols. Rat brain microsomes were also found to reduce saturated fatty acids ranging from 12 to 22 carbon atoms, and oleic acid to the corresponding alcohols in the presence of ATP, coenzyme A, Mg²⁺, and NADPH. Chain length selectivity in the reduction was less pronounced than that in alkyl DHAP synthesis. The data indicate that the alkyl and alk-1-enyl composition of rat brain ether lipids is controlled by substrate specificity in the formation of both fatty alcohol and alkyl dihydroxyacetone phosphate.     

Rapid synthesis and turnover of brain microsomal ether phospholipids in the adult rat.

The rates of synthesis, turnover, and half-lives were determined for brain microsomal ether phospholipids in the awake adult unanesthetized rat. A multicompartmental kinetic model of phospholipid metabolism, based on known pathways of synthesis, was applied to data generated by a 5 min intravenous infusion of [1,1-(3)H]hexadecanol. At 2 h post-infusion, 29%, 33%, and 31% of the total labeled brain phospholipid was found in the 1-O-alkyl-2-acyl-sn-glycero-3-phosphate, ethanolamine, and choline ether phospholipid fractions, respectively. Autoradiography and membrane fractionation showed that 3% of the net incorporated radiotracer was in myelin at 2 h, compared to 97% in gray matter microsomal and synaptosomal fractions. Based on evidence that ether phospholipid synthesis occurs in the microsomal membrane fraction, we calculated the synthesis rates of plasmanylcholine, plasmanylethanolamine, plasmenylethanolamine, and plasmenylcholine equal to 1.2, 9.3, 27.6, and 21.5 nmol. g(-1). min(-1), respectively. Therefore, 8% of the total brain ether phospholipids have half-lives of about 36.5, 26.7, 23.1, and 15.1 min, respectively. Furthermore, we clearly demonstrate that there are at least two pools of ether phospholipids in the adult rat brain. One is the static myelin pool with a slow rate of tracer incorporation and the other is a dynamic pool found in gray matter.The short half-lives of microsomal ether phospholipids and the rapid transfer to synaptosomes are consistent with evidence of the marked involvement of these lipids in brain signal transduction and synaptic function.     

Synthesis and evaluation of radioiodinated cholesteryl ethers as lipoprotein probes.

Two novel cholesteryl ether derivatives were synthesized and radioiodinated: (1) [125I]cholesteryl m-iodobenzyl ether (125I-CIBE) and (2) [125I]cholesteryl 12-(m-iodophenyl)dodecyl ether (125I-CIDE). These radioiodinated ethers were incorporated into low-density lipoprotein (LDL) by incubating the compounds (solubilized in saline with Tween-20) with isolated LDL or with whole plasma. Such LDL preparations were taken up by cultured fibroblasts in a receptor-dependent manner similar to that of radioiodinated LDL. Upon injection into guinea pigs, 125I-CIBE-labeled guinea pig LDL cleared from the plasma similarly to radioiodinated guinea pig LDL. The primary sites of 125I-CIBE uptake were the adrenal and the liver, and the compound was stable to both hydrolysis and deiodination over 24 h. In summary, 125I-CIBE and 125I-CIDE, like previously described tritiated cholesteryl ethers, appear to be potentially useful tracers of cholesteryl ester uptake. Moreover, these radioiodinated probes have the advantage of being more easily quantitated in tissue samples as well as being detectable by noninvasive scintigraphic imaging.     

Aldehydic peptides inhibiting renin

The metabolism of 20:4 (arachidonic acid) in alkenylacyl, alkylacyl and diacyl lipid classes in choline glycerophospholipids (CGP) and ethanolamine glycerophospholipids (EGP) in rabbit alveolar macrophages was examined. [3H]20:4 was very rapidly incorporated into diacyl glycerophosphocholine (GPC). After the removal of free 20:4, the radioactivity was gradually lost from diacyl GPC. Concomitantly, the radioactivities in alkylacyl GPC and alkenylacyl glycerophosphoethanolamine (GPE) were increased, indicating that 20:4 was mobilized from diacyl GPC to alkylacyl GPC and alkenylacyl GPE. The mobilization was considered to be a 20:4-specific event. The gradual accumulation of 20:4 in ether phospholipids leads to a high abundance of 20:4 in these lipids. These results suggest metabolic relationships between 20:4 and ether phospholipids, including platelet-activating factor (PAF).