Ether-linked Lipids Research Articles

Cellular morphogenesis in a halophilic archaebacterium

Cultures of a red halophilic archaebacterium exhibiting a complex morphology and cellular morphogenesis were obtained on a medium containingHalobacterium cutirubrum cell lysate. On primary culture the organism grew as an amorphous cellular mass 20 or more micrometers in diameter and underwent multiple internal cellular subdivision to produce a multicellular structure consisting of cuboidal cells of submicron dimensions. These disaggregated, elongated, cells became motile and multiplied by budding, thereby resembling the eubacteriumGeodermatophilus. The new isolates are identified as archaebacteria on the basis of their response to antibiotics, probable absence of peptidoglycan, and the presence of ether-linked lipids.

Quantitative analysis of ether-linked lipids as alkyl- and alk-1-enyl-glycerol benzoates by high-performance liquid chromatography

Methodology for the quantitative and qualitative analyses of alkyl- and alk-1-enyl-glycerols derived by Vitride reduction of ether-linked glycerolipids in the presence of an internal standard was developed. The procedure involved preparation of benzoate derivatives that were subsequently analyzed by high-performance liquid chromatography with detection at 228 nm. Separation of the glycerol ether dibenzoates on a C 18 reverse-phase column allowed for the simultaneous quantitation and the determination of chain length of both alkyl- and alk-1-enyl-glycerols in a single chromatographic run of less than 15 min. The method was accurate (<10% error), reproducible, and sensitive (<1 μg per component). Application of the method to the analysis of phospholipids from L-M cells grown in the presence and absence of elaidic acid demonstrated that the cells incorporated a portion of the trans acid supplement (presumably via the fatty alcohol) into the side chains of both alkyl- and alk-1-enyl-glycerol-containing phosphatides.

The interaction of dialkyl ether lecithins with phospholipase A2 (Naja naja naja).

Dialkyl lecithins are nonhydrolyzable substrate analogues for phospholipase A2 (Naja naja naja). Short chain dialkyl lecithins (which form monomers and micelles), and long chain ether-linked lecithins (which form bilayers or can be solubilized in detergent micelles) have been used to study phospholipase A2/phospholipid interactions. The results of gel filtration, 1H and 31P NMR spectroscopy, kinetic assays and UV difference spectroscopy show that binding of the ether-linked lecithins is independent of metal ion in pure lecithin systems (monomer, micelle, or bilayer), but requires Ca2+ for tight binding when Triton X-100 is used to solubilize the lecithin in mixed micelles. The apparent KD for dihexadecyl phosphatidylcholine is 2 mM in sonicated vesicles, but is increased to 6 mM in Triton-mixed micelles in the presence of Ca2+. When phospholipase A2 binds monomeric and micellar dialkyl lecithin, the lecithin phosphate resonance is broadened considerably, indicating that head group motion is restricted. Ca2+ enhances this interaction with the head group in these pure lipid systems. In similar 31P NMR experiments with dihexadecyl phosphatidylcholine solubilized in Triton X-100 mixed micelles, phospholipase A2 does not cause comparable broadening of the phosphate resonance, even in the presence of Ca2+ where gel filtration shows binding occurs. The kinetic effect of the ether-linked lipid inhibitors on phospholipase activity toward ester-linked lecithins is also modulated by the presence of Triton X-100. Ether-linked lecithins are much more potent inhibitors in two-component (dialkyl- diacyl-) lecithin micelles and bilayers than in complex mixed micelles with Triton X-100. Detergent-induced changes in the characteristics of the micellar interface are implicated in this inhibition phenomenon. The interaction of phospholipase A2 with sonicated vesicles of dihexadecyl phosphatidylcholine has also been examined by 1H and 31P NMR spectroscopy. When phospholipase A2 binds to vesicles, it promotes loss of encapsulated lanthanide ions, and appears to accelerate transbilayer phospholipid "flip-flop" rates. These results are discussed in terms of membrane asymmetry studies using phospholipase A2.

Comparative studies on the ether-linked lipids of ratfish and sharks-VI. Compositions of ether-linked lipids in livers of two species of ratfish.

The compositions of diacyl glyceryl ethers and neutral plasmalongens of the liver lipids of male and female specimens of two species of deep-sea ratfish Hydrolagus barbouri and Rhinochimaera pacifica were investigated. The livers of the examined ratfish yielded high lipid contents (75.9%-82.9%). Neutral lipids were the perdominant classes, accounting for more than 98% of the total lipid contents. The livers of these fish contained, in addition to 22.8%-31.7% triglycerides, ether-linked lipids of 53.5%-63.4% diacyl glyceryl ethers and 1.0%-6.7% neutral plasmalogens. In both species, the component alkyl glyceryl ethers of diacyl glyceryl ethers were found to consist mostly of monoenes (76.2%-81.1%) and saturates (15.5%-19.6%) of 14-24 carbon atoms for the alkyl moiety. The principal components found were: 18:1 (43.7%-57.5%), 16:1 (6.8%-13.5%), 20:1 (5.7%-8.7%), and 16:0 (5.4%-7.8%). The component aldehydes obtained from alk-l-enyl glyceryl ethers of neutral plasmalogens were mostly composed of monoenes (63.8%-81.8%) and saturates (18.8%-34.0%) of 14-24 carbon atoms. The major constituents were: 20:1 (40.3%-55.2%), 18:1 (5.4%-25-8%), 18:0 (5.7%-18.1%), and 16:0 (5.3%-8.0%). The component fatty acids of diacyl glyceryl ethers and neutral plasmalogens were roughly similar in composition to those of triglycerides. Monoenes having 18:1, 20:1, and 22:1 acids as the predominant components were found.

Formation of alkylacyl- and diacylglycerophosphocholines via diradylglycerol cholinephosphotransferase in rat liver

We found that diacylglycerol and alkylacylglycerol choline phosphotransferase activities exhibited similar pH optima, thermolabilities and inhibitions by Mn 2+, and dithiothreitol. The V max of diacylglycerol cholinephosphotransferase was higher (approx. 1–2-fold) than the V max of alkylacylglycerol cholinephosphotransferase. The K m value for diacylglycerol was somewhat greater than for the alkylacylglycerol, but no differences were found for the K m of CDPcholine with either type of diradylglycerol as the other substrate. Endogenous levels of diacylglycerols in microsomes were 24.3 nmol/mg protein, whereas no detectable amount of alkylacylglycerols was observed. Results from this study indicate that a similar or identical enzyme catalyzes the formation of 1,2-diacyl- and 1-alkyl-2-acyl- sn-glycero-3-phosphocholine and that the availability of diradylglycerols and the turnover rate of ether-linked lipids would appear to be important factors in controlling the level (under 1%) of 1-alkyl-2-acyl- sn-grycero-3-phosphocholine in rat liver.

Increasing the levels of ether-linked lipids in L-M cells by glyceryl ether supplementation depresses growth and choline utilization

To gain insight into the role of alkyl-linked lipids in biological systems, we added hexadecylglycerol (a precursor of complex ether-linked lipids) to medium required for the growth of L-M cells in culture. L-M fibroblasts cultured through several generations in the presence of hexadecylglycerol grow at a reduced rate. Experimental cells at their sixth passage, with 2 μg supplement/ml, double at 50% the rate of control cell populations. Hexadecylglycerol (10 μg/ml) added 1 day after cell passage does not retard growth; however, within 1 h it decreases the incorporation of choline into the choline glycerophosphatide fraction. Inhibition is specific for choline; ethanolamine incorporation is not affected. The inhibition of choline utilization by hexadecylglycerol-treated cells is dose-dependent and reaches a maximum 12 h after supplementation. Cellular uptake of choline is reduced (approx. 17%) but not as much as the incorporation of choline into the phospholipids (approx. 60% at 12 h). The assimilation of ether lipid precursor into cellular phospholipids was followed by incubating cells with [1- 14C]hexadecylglycerol. Incorporation of radioactivity into cellular phospholipids begins to plateau after 24 h, whereas the interference of hexadecylglycerol with choline metabolism could be detected as early as 1 h. The majority of the radioactivity recovered from cells incubated with labeled hexadecylglycerol is localized in the microsomal fraction (56%), where the label was distributed as free hexadecylglycerol, alkylacyl-phospholipids and alkyldiacylglycerols. These results show that the supplementation of a glyceryl ether to L-M fibroblast growth media selectively inhibits the utilization of choline for choline glycerophospholipid biosynthesis and causes a reduction in cell growth rate when cells are continually passaged in the presence of the glyceryl ether.

Ether-linked lipids of Balb/c3T3, SV3T3 and concanavalin A-selected SV3T3 revertant cells

Ether-linked lipids were analyzed in Balb/c3T3, SV3T3 and Concanavalin A-selected SV3T3 revertant cells. The three cell lines were found to contain significant quantities of alk-1-enyl- and alkyl-linked phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and small amounts of alkyldiacylglycerols. Compared to 3T3 cells, SV3T3 cells contain a higher amount of alk-1-enyl-linked PC, while in SV3T3 revertant cells the concentrations of the various ether lipids are similar to those of 3T3 cells. The major difference in the composition of ether groups of SV3T3 cells, compared to 3T3 cells, is an increase of 18:0 accompanied by a decrease of 18:1 in the alk-1-enyl-linked PE and PC. Alk-1-enyl-linked PC of SV3T3 revertant cells also shows an increase of 18:0, while the decrease of 18:1 was not statistically significant.

Ether lipid studies in mouse C3H/10T1/2 cells and a 3-methylcholanthrene-transformed clone

C3H/10T1/2 mouse embryo cells and a transformed clone were used in these initial experiments to investigate the future application of this model culture system to studies of ether-linked lipids in cancer cells. Clone 8 cells are nontumorigenic, nontransformed, and maintain normal morphology to passages 15–20. Clone 16 cells were derived from morphologically transformed foci of clone 8 cells exposed to the chemical carcinogen, 3-methylcholanthrene, and are highly tumorigenic. The data presented here demonstrate that the high amounts of ether-linked lipids, characteristic of tumors, are likewise elevated in cells that have been oncogenically transformed in vitro. When incubated with labeled fatty alcohols, the transformed cells show a stimulated incorporation of radioactivity into alkyldiacylglycerols (>100% over clone 8), whereas radioactivity in the alkyl moiety of the phospholipids is not altered. Analysis of the lipids formed from [1- 14C]hexadecanol indicates that the nontransformed cells have a greater capacity to oxidize hexadecanol and incorporate the resulting carboxylic acid into acyl groups. Quantitative analysis of cellular lipids shows that in the oncogenically transformed cells alkyldiacylglycerols are increased (123% over clone 8).

Comparative studies on the ether-linked lipids of ratfish and shark-III. Distribution of squalene and diacyl glyceryl ethers in the different tissues of deep-sea shark, Dalatias licha.

The lipid compositions of the different tissues and gut content of deep-sea shark, Dalatias licha, were determined. In this species, the liver contained 83.4% lipids, which was much larger than that of muscle, ovary and gut content, ranging from 17.3% to 0.8%. The liver and ovary lipids were characterized by high percentages of squalene (40.1%, 20.3%) and diacyl glyceryl ethers (27.8%, 35.7%). On the contrary, the lipids of muscle and gut content contained small amounts of the above types of lipids. Glyceryl ethers of diacyl glyceryl ethers of liver and ovary were very similar in composition: the predominant constituents were 18:1 (52.7%-50.8%) and 20:1 (27.1%-24.7%). In both liver and ovary, the component fatty acids of diacyl glyceryl ethers and triglycerides were also similar in composition, predominantly consisting of monoenes (74.8%-81.3%). Based on the characteristic lipid composition and/or the component glyceryl ethers or fatty acids of diacyl glyceryl ethers and triglycerides, it is suggested that the ovary tissue has directly accumulated the liver lipids for egg development.

Activities of enzymes involved in the metabolism of ether-linked lipids in normal and neoplastic tissues of rat

We have compared the activities of three enzymes (acyl-CoA reductase, fatty alcohol:NAD + oxidoreductase, and alkyl synthase) involved in the metabolism of ether lipids in rat liver and two tumors that contained various levels of ether lipids. The activity of fatty alcohol: NAD + oxidoreductase was high and the activities of acyl-CoA reductase and alkyldihydroxyacetone-P synthase were low in liver, which has low amounts of ether glycerolipids. On the other hand in Fischer R-3259 sarcoma, where high concentrations of ether-linked lipids were present, the reverse pattern was observed. In Morris hepatomas 5123C, with an intermediate value of ether lipids, the activities of all three enzymes ranged between that found in liver and Fischer sarcomas.

Lipid composition of yoshida ascites hepatoma and of livers and blood plasma from host and normal rats

The lipid composition of Yoshida ascites hepatoma cells was analyzed together with that of ascitic plasma and of livers and blood plasma from host and normal rats. In comparison to normal livers, host livers showed no significant differences in the content of the various lipid classes, but contained a higher percentage of palmitic acid and a lower proportion of arachidonic acid in the major phospholipid classes. In addition, tumor growth induced a marked hypertriglyceridemia in host animals; changes in the concentration of other plasma lipid classes were not statistically significant. The ascitic plasma contained small amounts of lipids mainly constituted by cholesteryl esters and phospholipids. Yoshida hepatoma cells contained less phospholipids in comparison to both host and normal liver, while the increased level of triglycerides and the decrease of free fatty acids were not statistically significant. Hepatoma cells showed appreciable amounts of ether-linked lipids associated in part to neutral lipids (as glyceryl ether diesters) and, in part, to ethanolamine and choline phosphoglycerides. The alkyl groups in GEDE as well as in ethanolamine and choline phosphoglycerides were mainly constituted by C16:0 and C18:0 followed by C18:1. The alk-1-enyl groups in ethanolamine and choline phosphoglycerides were C16:0 and C18:0 with only a minor proportion of C18:1. In comparison to both host and normal liver, Yoshida hepatoma cells showed significant changes in the fatty acid composition of neutral lipids and phospholipids. Some of the major changes consisted of an increase of monoenoic acids associated with a decrease of arachidonic and docosahexaenoic acids in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol.

Effects of phospholipid base analogues on the subcellular membrane ether composition of suspension cultured LM cells

A strain of mouse fibroblasts, LM cells, was cultured in suspension with a chemically defined medium in the absence of lipid or serum. The subcellular distribution of ether-linked lipids in these cells was determined. O-alkyl and O-alk-1-enyl glycerolipids were found in plasma membranes, microsomes, and mitochondria. The glyceryl ether diesters from all three membrane fractions were devoid of O-alk-1-enyl moieties. Phospholipids contained both O-alkyl and O-alk-1-enyl ether chains. The distribution of these lipids among the subcellular fractions was not uniform. Microsomes contained the largest quantities of O-alkyldiacylglycerols, O-alkylacylphosphatides, and O-alk-1-enylacylphosphatides. Mitochondria were essentially devoid of O-alk-1-enyl phospholipids. The in vivo incorporation of new polar head groups such as N, N-dimethyl-ethanolamine, N-monomethylethanolamine, or ethanolamine instead of N, N, N-trimethylethanolamine (choline) into LM suspension cell membrane phospholipids resulted in increased levels of ether-linked glycerolipids. These increases were inversely proportional to the number of CH 3 groups on the nitrogen atom of the base supplement. Both neutral lipids and phospholipids contained increased (2-fold or greater) quantities of O-alkyl moieties, but the levels of O-alk-1-enyl ether moieties in the phospholipids were slightly decreased or unchanged. These alterations in ether lipid content occurred primarily in the microsomes and plasma membranes while mitochondrial ether content or composition was not altered significantly. The chain length composition and degree of unsaturation of the O-alkyl and O-alk-1-enyl side chains from neutral lipids and phospholipids of these membranes were determined. Small (less than 10%) increases in unsaturation of O-alkyl side chains and larger (2-fold) increases in unsaturation of O-alk-1-enyl side chains of plasma membranes and microsomes were noted. These changes were not consistent with developmental alterations during the growth phase since the degree of unsaturation of ether-linked lipids from control LM cells decreased with age of the culture, an affect opposite to that noted in the presence of base analogues. In addition, the increases in levels of ether lipids after base analogue supplementation were much greater than could be accounted for simply by alterations in growth phase of LM suspension cells.

Confirmation of biosynthesis and partial structural characterization of the ether-linked ethanolamine phosphoglycerides of Heliothis

Tritium incorporation in vivo in the larval and pupal tobacco budworm from 3H-1-octadecanol resulted in high specific activities of ether-linked lipids but very low labeling of acyl moieties. These observations are confirmatory evidence for the biosynthesis of alkyl and alk-1-enyl glyceryl ethers in this insect. Analysis of the labeled ethers on arsenite- and borate-impregnated thin-layer chromatography plates, and derivatives confirms their identity and shows that the ether bond is attached only to the sn-1 position of glycerol.

A Lysophospholipase D Pathway in the Metabolism of Ether-linked Lipids in Brain Microsomes

Abstract Microsomal preparations from rat brain in the presence of Mg2+ hydrolyzed the ethanolamine, choline, and phosphate moieties from 1-[1-14C]hexadecyl-sn-glycero-3-phosphorylethanolamine, 1-[1-14C]hexadec-1-enyl-sn-glycero-3-phosphorylethanolamine, and 1-[1-14C]hexadecyl-sn-glycero-3-phosphorylcholine. Studies of the hydrolysis of 1-[1-14C]-hexadecyl-sn-glycero-3-phosphorylethanolamine in this system indicated that the ethanolamine moiety was first removed by a phosphodiesterase to form 1-[1-14C]hexadecylglycero-3-phosphate which was then dephosphorylated to form 1-[1-14C]hexadecylglycerol. Only minimal hydrolysis occurred when the 2-positions of the substrates were acylated; otherwise the phosphodiesterase activity was similar to that of phospholipase D from plants. The occurrence of such a lysophospholipase D pathway has not been previously reported. When Ca2+ (5 mm) was added to the system instead of Mg2+ (5 mm), little, if any, stimulation occurred; higher concentrations of Ca2+ (25 mm) inhibited the reaction. Therefore, this reaction does not appear to be related to the Ca2+-stimulated base exchange reaction found in the brain by others.

Enzymatic Systems That Synthesize and Degrade Glycerolipids Possessing

Publisher Summary Ether bonds in glycerolipids were first detected in biological materials. The O-alkyl linkage was found in marine organisms, whereas the O-alk-1-enyl linkage was first detected in mammalian cells. This chapter discusses the nomenclature and illustrates the structures of known O-alkyl and O-alk-1-enyl lipid classes that are of biological interest. It presents the conclusions drawn from enzyme studies on the biosynthesis and degradation of alkyl and alk-1-enyl bonds in glycerolipids and their precursors; in vivo results and chemical syntheses have been discussed. The function of the ether-linked lipids in biological systems is unknown, although their structural role in membranes, especially surface membranes, has become apparent in a number of investigations. Chromatographic isolation procedures and subsequent identifications in biochemical investigations, as well as chemical syntheses, have led to the discovery of a large number of distinct ether-linked lipid classes. The lipid classes that contain ether bonds are structural analogs of the acylated lipid classes.

Ether-containing lipids of the slime mold,Physarum polycephalum: I. Characterization and quantification.

Rapidly growing plasmodia of the acellular slime mold,Physarum polycephalum, contain large amounts of ether-linked lipids. The ether bonds occur principally in the phospholipids, where plasmalogens account for 21-24 mole per cent of the total and alkyl ether phospholipids comprise 12 mole per cent of the total. Plasmalogens account for over half of the ethanolamine phosphatides, while the alkyl ether derivatives are more uniformly distributed among the various phospholipid fractions. The 16 carbon side chain is by far the major component of both the saturated and the α,β-unsaturated ether derivatives. The nature and amounts of ether lipids present inPhysarum add to the growing evidence that Myxomycetes are more closely related to protozoa than to fungi.

Ester and ether-linked lipids in the mandibular canal of a porpoise (Phocoena phocoena). Occurrence of isovaleric acid in glycerolipids.

Ester and ether-linked lipids in the mandibular canal of a porpoise (Phocoena phocoena). Occurrence of isovaleric acid in glycerolipids.

Tumor lipids: Characterization of the lipids isolated from membranous material

Membranous material was prepared from Ehrlich ascites carcinoma cells by zonal centrifugation. The distribution of fatty acids among the individual neutral lipid and phospholipid classes isolated from the membrane preparation was determined. Survey electron microscopy indicated that the preparations consisted of vesicles of rough surfaced endoplasmic reticulum, lysosomal-like structures, virus-like particles, few intact mitochondria, and unidentified amorphous material, in addition to plasma membranes. Lipids and proteins accounted for approximately one-third and two-thirds of the membranous material. Phospholipids accounted for two-thirds of the total lipids. The membranous material contained higher levels of free fatty acids, cholesterol, diacyl phosphatidyl choline, diacyl phosphatidyl ethanolamine, and lower levels of glyceryl ether diesters and cholesterol esters than whole cells. The fatty acid composition of all the lipid classes isolated from the membranous material, except phosphatidyl inositol and cholesterol esters, was similar to the fatty acid distribution of the corresponding classes obtained from whole cells. Cholesterol esters contained elevated levels of 16:1 and stearic acid accounted for 88% of phosphatidyl inositol fatty acids. Phosphatidyl choline and phosphatidyl ethanolamine isolated from the membranous preparations contained 1% or less alkyl acyl phosphatides, and plasmalogens were not detected. The lipids of whole Ehrlich ascites cells have been shown to contain high levels of ether-linked lipids, whereas these data indicate that alkyl and alk-1-enyl glyceryl ethers are virtually absent from membrane preparations of these cells. These results indicate that the ether-linked lipids are not distributed equally among all cell structures.

The biosynthesis of alkyl ether bonds in lipids by a cell-free system

Investigators have found the biological significance and the biosynthetic pathways of ether-linked lipids containing glycerol difficult to determine (1). Despite available knowledge concerning enzymes for the biocleavage (2,3), deacylation (4), acylation (5,6), and phosphorylation (7) of glyceryl ethers, the biosynthesis of the ether bond in these compounds has not previously been demonstrated in a cell-free system. This note describes an active enzyme complex capable of converting intact l- 14C-labeled fatty alcohols into alkyl glyceryl ethers. The reaction has been demonstrated in whole homogenates and in microsomal-plus-supernatant fractions of transplantable preputial tumors in mice. These tumors are typical of neoplasms containing high quantities of alkyl ether linkages in both neutral- and phospho-glycerides (8).