Exogenous Ethanolamine Research Articles

Identification of the substrate‐binding sites in the phosphatidylserine synthase from Candida albicans

Systemic infections by Candida spp. have high mortality rates, and this is related, in part, to limitations in current antifungals. Thus, there is a need for novel antifungals. The phosphatidylserine (PS) synthase (annotated Cho1) from C. albicans has been identified as a potential drug target because [1] it is required for virulence in mouse models of Candidiasis, [2] it is absent in humans, and [3] it is highly conserved among fungal pathogens. Inhibitors of Cho1 could be used as lead compounds for drug development. Rational drug design is one way to develop specific inhibitors, but neither the substrate-enzyme interactions nor the substrate-binding sites have been well-defined for Cho1. Therefore, we aim to biochemically identify and characterize the substrate-binding sites of Cho1, which binds cytidine-diphosphate-diacylglycerol (CDP-DAG) and serine, and catalyzes their condensation to form PS. For the CDP-DAG-binding site, a conserved CDP-alcohol-binding (CAPT) motif is present in Cho1. We tested this site for its role in PS synthesis by mutating the conserved residues using standard alanine-scanning mutagenesis, and using two assays to measure the impact of these mutations on PS synthesis. The first assay measures the impact of PS synthesis in whole cells based on their growth. PS is used to synthesize phosphatidylethanolamine (PE), and in the absence of PS, C. albicans must acquire exogenous ethanolamine to make PE by a salvage pathway. Thus, mutants lacking PS synthase activity grow very poorly in media lacking ethanolamine. The second assay measures PS synthesis directly in crude membranes based on incorporation of [3H]serine into [3H]PS. These experiments revealed that, with the exception of one residue (R133), all alanine substitutions of the conserved amino acids within the fungal CAPT motif showed decreased Cho1 function. A subsequent R133E mutant displayed reduced activity compared to R133A, indicating residue R133 interacts with CDP-DAG in a dispensable manner. For the serine-binding site, by using computational tools, we predicted potential serine-binding residues. Using alanine-scanning mutagenesis, we found that some of these predicted residues are required for Cho1 function. Two of the alanine substitution mutants, L184A and R189A, were characterized regarding their Michaelis-Menten kinetics. The mutant L184A, which displayed enhanced activity both for growth and in vitro PS synthase activity, showed an increased Vmax and an unchanged Km. The gain-of-function mechanism of this mutation is currently unknown. On the contrary, R189A, which showed decreased growth and in vitro PS synthase activity, increased the Km for serine but not Vmax, indicating that the residue R189 is involved in serine-binding. We plan to continue to determine if other potential residues directly bind serine, as this could be useful in future rational inhibitor design studies.

High-throughput screening for phosphatidylserine decarboxylase inhibitors using a distyrylbenzene-bis-aldehyde (DSB-3)-based fluorescence assay

Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity in vitro are not suitable for high-throughput drug screening. The finding that the PkPSD from Plasmodium knowlesi can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity in vitro have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC50 values ranging from 3.1 to 42.3 μm Lead compounds were evaluated against the pathogenic yeast Candida albicans in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native C. albicans PSD mitochondrial activity and C. albicans growth, with an MIC50 of 22.5 and 15 μg/ml without ethanolamine and an MIC50 of 75 and 60 μg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.

Overproduction of Phospholipids by the Kennedy Pathway Leads to Hypervirulence in Candida albicans.

Candida albicans is an opportunistic human fungal pathogen that causes life-threatening systemic infections, as well as oral mucosal infections. Phospholipids are crucial for pathogenesis in C. albicans, as disruption of phosphatidylserine (PS) and phosphatidylethanolamine (PE) biosynthesis within the cytidine diphosphate diacylglycerol (CDP-DAG) pathway causes avirulence in a mouse model of systemic infection. The synthesis of PE by this pathway plays a crucial role in virulence, but it was unknown if downstream conversion of PE to phosphatidylcholine (PC) is required for pathogenicity. Therefore, the enzymes responsible for methylating PE to PC, Pem1 and Pem2, were disrupted. The resulting pem1Δ/Δ pem2Δ/Δ mutant was not less virulent in mice, but rather hypervirulent. Since the pem1Δ/Δ pem2Δ/Δ mutant accumulated PE, this led to the hypothesis that increased PE synthesis increases virulence. To test this, the alternative Kennedy pathway for PE/PC synthesis was exploited. This pathway makes PE and PC from exogenous ethanolamine and choline, respectively, using three enzymatic steps. In contrast to Saccharomyces cerevisiae, C. albicans was found to use one enzyme, Ept1, for the final enzymatic step (ethanolamine/cholinephosphotransferase) that generates both PE and PC. EPT1 was overexpressed, which resulted in increases in both PE and PC synthesis. Moreover, the EPT1 overexpression strain is hypervirulent in mice and causes them to succumb to system infection more rapidly than wild-type. In contrast, disruption of EPT1 causes loss of PE and PC synthesis by the Kennedy pathway, and decreased kidney fungal burden during the mouse systemic infection model, indicating a mild loss of virulence. In addition, the ept1Δ/Δ mutant exhibits decreased cytotoxicity against oral epithelial cells in vitro, whereas the EPT1 overexpression strain exhibits increased cytotoxicity. Taken altogether, our data indicate that mutations that result in increased PE synthesis cause greater virulence and mutations that decrease PE synthesis attenuate virulence.

Metabolome analysis reveals ethanolamine as potential marker for improving lipid accumulation of model photosynthetic organisms

AbstractBACKGROUND: Many researchers focus on exploring approaches to improve lipid productivity by photosynthetic organisms. Metabolomic analysis provides a new way to globally explore metabolic changes. To elevate lipid yields in the process of biodiesel production using photosynthetic organisms as feedstock, metabolomics combined with orthogonal partial least‐squares discriminant analysis (OPLS‐DA) is being applied widely to seek potential markers in regulation of lipid production. Herein, metabolic differences among three photosynthetic organisms Scenedesmus obliquus, Synechocystis sp. PCC6803 and Anabaena sp. PCC7120 were analyzed by gas chromatography coupled with time‐of‐flight mass spectrometry (GC‐TOF‐MS).RESULTS: In total, 74 metabolites were identified by GC‐TOF‐MS. The OPLS‐DA model revealed good correlation between metabolites and lipid content (R2 = 0.9916). Nine compounds including ethanolamine were selected as potential markers affecting lipid accumulation. Herein, the highest level of C18:3 revealed its roles in regulation of the unsaturated degree of fatty acids. Further study revealed that exogenous ethanolamine (2 mmol L−1) significantly increased the lipid content (22%) in Scenedesmus obliquus. In particular, ethanolamine significantly elevated the levels of C16:2, C18:1, and C18:2.CONCLUSION: Metabolomic analysis is a useful tool to search for potential markers to improve lipid accumulation of photosynthetic organisms. Ethanolamine can be regarded as an alternative compound to improve lipid content and fatty acid levels for biodiesel production. Copyright © 2012 Society of Chemical Industry

Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae

In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[(3)H(G)]serine and [(14)C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of "correct" PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.

Placental Thrombosis and Spontaneous Fetal Death in Mice Deficient in Ethanolamine Kinase 2

Ethanolamine kinase catalyzes the first step in the CDP-ethanolamine pathway for the formation of the major membrane phospholipid phosphatidylethanolamine (PtdEtn). In this work, the predicted Etnk2 cDNA was established as a soluble protein with ethanolamine-specific kinase activity that was most highly expressed in liver. Mice with an inactivated Etnk2 gene were derived, and its absence reduced the rate of PtdEtn synthesis from exogenous ethanolamine in hepatocytes. PtdEtn is a major precursor to phosphatidylcholine in liver; however, Etnk2(-/-) mice did not have reduced amounts of either PtdEtn or phosphatidylcholine or an altered phospholipid molecular species distribution. The knock-out animals were able to adapt to a choline-deficient diet. The Etnk2(-/-) mice exhibited a maternal-specific intrauterine growth retardation phenotype that resulted in a 33% reduction in litter size and frequent perinatal death. Histological analysis of pregnant Etnk2(-/-) females showed that fetal development failed at the late stage of pregnancy in a significant percentage of embryos because of the appearance of extensive placental thrombosis. These results illustrate a non-redundant role for EtnK2 expression in regulating placental hemostasis.

Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine biosynthetic machinery with the prohibitin complex of Saccharomyces cerevisiae.

The majority of mitochondrial phosphatidylethanolamine (PtdEtn), a phospholipid essential for aerobic growth of yeast cells, is synthesized by phosphatidylserine decarboxylase 1 (Psd1p) in the inner mitochondrial membrane (IMM). To identify components that become essential when the level of mitochondrial PtdEtn is decreased, we screened for mutants that are synthetically lethal with a temperature-sensitive (ts) allele of PSD1. This screen unveiled mutations in PHB1 and PHB2 encoding the two subunits of the prohibitin complex, which is located to the IMM and required for the stability of mitochondrially encoded proteins. Deletion of PHB1 and PHB2 resulted in an increase of mitochondrial PtdEtn at 30 degrees C. On glucose media, phb1Delta psd1Delta and phb2Delta psd1Delta double mutants were rescued only for a limited number of generations by exogenous ethanolamine, indicating that a decrease of the PtdEtn level is detrimental for prohibitin mutants. Similar to phb mutants, deletion of PSD1 destabilizes polypeptides encoded by the mitochondrial genome. In a phb1Delta phb2Delta psd1(ts) strain the destabilizing effect is dramatically enhanced. In addition, the mitochondrial genome is lost in this triple mutant, and nuclear-encoded proteins of the IMM are assembled at a very low rate. At the nonpermissive temperature mitochondria of phb1Delta phb2Delta psd1(ts) were fragmented and aggregated. In conclusion, destabilizing effects triggered by low levels of mitochondrial PtdEtn seem to account for synthetic lethality of psd1Delta with phb mutants.

Construction of phosphatidylethanolamine-less strain of Saccharomyces cerevisiae. Effect on amino acid transport.

A triple yeast mutant was constructed which lacks BST1, the gene for sphingosine lyase, besides the phosphatidylserine decarboxylases PSD1 and PSD2. In this yeast mutant, which can only be grown in the presence of exogenous ethanolamine, phosphatidylethanolamine can be depleted to very low levels. Under those conditions, respiration as well as glucose and 3-O-methylglucose uptake proceed unaffected. Plasma membrane ATPase is as active in these cells as that of control cells grown in the presence of ethanolamine. Drastically decreased, however, are H+/amino acid symporters. The activities of arginine (Can1p), proline (Put4p) and general amino acid permease (Gap1p) are decreased more than 20-fold. Amino acid transport in yeast is dependent on coupling to the proton motive force. It can be envisaged that phosphatidylethanolamine might play a role in this process or in the early steps of the secretion pathway common for all amino acid permeases or, eventually, it could affect the transport proteins directly at the plasma membrane Transformation of the triple mutant with a CEN plasmid harbouring BST1 wild-type gene totally reversed its phenotype to that observed in the double mutant.

Biosynthesis and turnover of anandamide and other N-acylethanolamines in peritoneal macrophages

Polyunsaturated N-acylethanolamines (NAEs), including anandamide (20:4 n−6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca 2+ ionophore A23187 had no effect on the production of NAE or its precursor N-acylphosphatidylethanolamine ( N-acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N-acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of 18O from H 2 18O-containing media into the amide carbonyls of both NAE and N-acyl PE demonstrated a rapid, constitutive turnover of both lipids.

Chronic ethanol increases the cannabinoid receptor agonist anandamide and its precursor N-arachidonoylphosphatidylethanolamine in SK-N-SH cells.

In an earlier study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane. In the present study, we investigated the effect of chronic EtOH on the formation of anandamide (AnNH), an endogenous cannabimimetic compound, and its precursor N-arachidonoylphosphatidylethanolamine (N-ArPE) in SK-N-SH cells that were prelabeled with [3H]arachidonic acid. The results indicate that exposure of SK-N-SH cells to EtOH (100 mM) for 72 h significantly increased levels of [3H]AnNH and [3H]N-ArPE (p < 0.05) (1.43-fold for [3H]AnNH and 1.65-fold for [3H]N-ArPE). Exposure of SK-N-SH cells to EtOH (100 mM, 24 h) inhibited initially the formation of [3H]AnNH at 24 h, followed by a progressive increase, reaching a statistical significance level at 72 h (p < 0.05). [3H]N-ArPE increased gradually to a statistically significant level after 48 and 72 h (p < 0.05). Incubation with exogenous ethanolamine (7 mM) and EtOH (100 mM, 72 h) did not result in an additive increase in the formation of [3H]AnNH. The formation of [3H]AnNH and [3H]N-ArPE by EtOH was enhanced by the Ca2+ ionophore A23187 or by the depolarizing agent veratridine and the K+ channel blocker 4-aminopyridine. Further, the EtOH-induced formation of [3H]AnNH and [3H]N-ArPE was inhibited by exogenous AnNH, whereas only [3H]AnNH formation was inhibited by the CB1 receptor antagonist SR141716A and pertussis toxin, suggesting that the CB1 receptor and G(i/o) protein mediated the regulation of AnNH levels. The observed increase in the levels of these lipids in SK-N-SH cells may be a mechanism for neuronal adaptation and may serve as a compensatory mechanism to counteract the continuous presence of EtOH. The present observation taken together with our previous results indicate the involvement of the endocannabinoid system in mediating some of the pharmacological actions of EtOH and may constitute part of a common brain pathway mediating reinforcement of drugs of abuse including EtOH.

Cloning and Expression of Mouse Liver Phosphatidylserine Synthase-1 cDNA: OVEREXPRESSION IN RAT HEPATOMA CELLS INHIBITS THE CDP-ETHANOLAMINE PATHWAY FOR PHOSPHATIDYLETHANOLAMINE BIOSYNTHESIS

In eukaryotic cells, phosphatidylserine (PtdSer) is synthesized by two distinct synthases on the endoplasmic reticulum by a base-exchange reaction in which the polar head-group of an existing phospholipid is replaced with serine. We report the cloning and expression of a cDNA for mouse liver PtdSer synthase-1. The deduced protein sequence is >90% identical to that of PtdSer synthase-1 from Chinese hamster ovary cells and a sequence from a human myeloblast cell line. PtdSer synthase-1 cDNA was stably expressed in M.9.1.1 cells which are mutant Chinese hamster ovary cells defective in PtdSer synthase-1 activity, are ethanolamine auxotrophs, and have a reduced content of PtdSer and phosphatidylethanolamine (PtdEtn). The growth defect of M.9.1.1 cells was eliminated, and a normal phospholipid composition was restored in the absence of exogenous ethanolamine, implying that the cloned cDNA encoded PtdSer synthase. Mouse liver PtdSer synthase-1 was also expressed in McArdle 7777 rat hepatoma cells. In addition to a 3-fold higher in vitro serine-exchange activity, these cells also exhibited enhanced choline- and ethanolamine-exchange activities and incorporated more [3H]serine into PtdSer than did control cells. However, the levels of PtdSer and PtdEtn in cells overexpressing PtdSer synthase-1 activity were not increased. Excess PtdSer produced by the transfected cells was rapidly decarboxylated to PtdEtn and the degradation of PtdSer, and/or PtdEtn derived from PtdSer, was increased. Moreover, the CDP-ethanolamine pathway for PtdEtn biosynthesis was inhibited. These data suggest that (i) cellular levels of PtdSer and PtdEtn are tightly controlled, and (ii) the metabolism of PtdSer and PtdEtn is coordinately regulated to maintain phospholipid homeostasis.

Evidence for an ethanolamine cycle: differential recycling of the ethanolamine moiety of phosphatidylethanolamine derived from phosphatidylserine and ethanolamine.

Evidence is presented for the operation of an ethanolamine-phosphatidylethanolamine (PtdEtn) cycle in Chinese hamster ovary cells. PtdEtn was labelled with [3H]ethanolamine and radioactivity was chased by incubation with 1 mM unlabelled ethanolamine. Radioactivity in [3H]PtdEtn gradually declined over a 23 h time period. In contrast, when the cells were incubated in medium lacking unlabelled ethanolamine, radioactivity in PtdEtn remained constant for at least 23 h. These observations suggest that the ethanolamine moiety is continuously released from PtdEtn and recycled back into PtdEtn. In cells incubated without unlabelled ethanolamine, labelled ethanolamine released from PtdEtn is re-incorporated into PtdEtn without significant dilution. In contrast, in cells incubated with unlabelled ethanolamine the specific radioactivity of the intracellular ethanolamine pool decreases as a result of dilution by the exogenous ethanolamine, hence radioactivity in PtdEtn gradually declines. Similar results were obtained for confluent and non-confluent cells. Our data also demonstrate that when PtdEtn is derived from phosphatidylserine decarboxylation, the ethanolamine cycle operates only in actively dividing, and not in confluent, cells, implying that PtdEtn derived from different biosynthetic origins [i.e. from decarboxylation of phosphatidylserine or from ethanolamine (most likely via the CDP-ethanolamine pathway)] is metabolized differently.

Identification of a Non-mitochondrial Phosphatidylserine Decarboxylase Activity (PSD2) in the Yeast Saccharomyces cerevisiae

Phosphatidylserine decarboxylase (PSD1) plays a central role in the biosynthesis of aminophospholipids in both prokaryotes and eukaryotes by catalyzing the synthesis of phosphatidylethanolamine. Recent reports (Trotter, P. J., Pedretti, J., and Voelker, D. R. (1993) J. Biol. Chem. 268, 21416-21424; Clancey, C. J., Chang, S.-C., and Dowhan, W. (1993) J. Biol. Chem. 268, 24580-24590) described the cloning of a yeast structural gene for this enzyme (PSD1) and the creation of the null allele. Based on the phenotype of strains containing a null allele for PSD1 (psd1-delta 1::TRP1) it was hypothesized that yeast have a second phosphatidylserine decarboxylase. The present studies demonstrate the presence of a second enzyme activity (denoted PSD2), which, depending on the method of evaluation, accounts for 4-12% of the total cellular phosphatidylserine decarboxylase activity found in wild type. Recessive mutations resulting in loss of this enzyme activity (denoted psd2) in cells containing the psd1-delta 1::TRP1 null allele also result in ethanolamine auxotrophy. When incubated with [3H]serine these double mutants accumulate label in phosphatidylserine, while very little (< 5%) is converted to phosphatidylethanolamine. In addition, these mutants have a approximately 70% decrease in the amount of total phosphatidylethanolamine even when grown in the presence of exogenous ethanolamine. Strains containing psd1 or psd2 mutations were utilized for the subcellular localization of the PSD2 enzyme activity. Unlike the PSD1 activity, the PSD2 enzyme activity does not localize to the mitochondria, but to a low density subcellular compartment with fractionation properties similar to both vacuoles and Golgi.

Limited metabolic interaction of serine with ethanolamine and choline in the turnover of phosphatidylserine, phosphatidylethanolamine and plasmalogens in cultured glioma cells

Modulation of choline phosphoglyceride turnover has been investigated extensively but less is known about regulation of serine and ethanolamine phosphoglyceride synthesis and turnover. We investigated incorporation and interactions of [ 3H(G)] l-serine, [1,2- 14C]ethanolamine and [ methyl- 3H]choline in cultured glioma cells. Exogenous serine did not compete with ethanolamine or choline incorporation and did not chase labeled headgroup from ethanolamine phosphoglycerides (PE); serine displaced headgroup of prelabeled phosphatidylserine (PtdSer) resulting in less labeled PtdSer for decarboxylation. In contrast, exogenous ethanolamine markedly chased labeled headgroup of non-plasmenylethanolamine phosphoglycerides (NP-PE) with less effect on plasmalogen (1- O-alk-1'-enyl-2-acyl- sn-glycero-3-phosphoethanolamine) whether headgroup was derived from [ 3H]serine or [ 14C]ethanolamine. Label in chase medium was mainly ethanolamine to 12 h; phosphoethanolamine was present with longer chase (12–48 h). Choline did not compete with serine incorporation and had little chase effect on PtdSer and PE. Choline and ethanolamine competitively interacted with preference for choline. These data suggest that (1) PtdSer synthesis in cultured glioma cells may involve more than headgroup exchange; (2) PE turnover with metabolite release to medium may involve both phospholipase D and phospholipase C; (3) acceleration of PE turnover by exogenous ethanolamine primarily involves NP-PE with lesser involvement of plasmalogen; and (4) in contrast to lack of interaction between serine and other headgroup precursors, choline and ethanolamine compete primarily at uptake.

Modulation of phosphatidylethanolamine biosynthesis by exogenous ethanolamine and analogues in the hamster heart.

In the hamster heart, exogenous ethanolamine is taken up by the heart and utilized for the biosynthesis of phosphatidylethanolamine. The role of the exogenous supply of ethanolamine on phosphatidylethanolamine biosynthesis was examined by perfusing hamster heart with various concentrations of labelled ethanolamine. Analysis of the radioactivity distributed in the ethanolamine-containing metabolites indicated that at low exogenous ethanolamine concentrations (< or = 0.1 microM), the conversion of phosphoethanolamine to CDP-ethanolamine was rate-limiting for phosphatidylethanolamine biosynthesis. However, perfusion with higher concentrations of ethanolamine (> or = 0.4 microM) resulted in the phosphorylation of ethanolamine becoming rate-limiting. Since the intracellular ethanolamine levels remained unchanged, the alterations in radioactivity distribution suggested that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis. The effects of ethanolamine analogues on ethanolamine uptake and subsequent conversion to phosphatidylethanolamine at physiological concentrations of exogenous ethanolamine were examined. Monomethylethanolamine was found to inhibit ethanolamine uptake, the conversion of ethanolamine to phosphoethanolamine and incorporation of radioactivity into phosphatidylethanolamine. The accumulation of radioactivity in the ethanolamine fraction by monomethylethanolamine, despite of the inhibition of ethanolamine uptake, further confirms the rate-limiting role of ethanolamine kinase in the biosynthesis of phosphatidylethanolamine.

Newly imported ethanolamine is preferentially utilized for phosphatidylethanolamine biosynthesis in the hamster heart

The effects of exogenous ethanolamine concentrations on ethanolamine uptake and its subsequent incorporation into phosphatidylethanolamine were examined. Hamster hearts were perfused with 0.04–1000 μM labelled ethanolamine. Analysis of radioactivity distribution in ethanolamine-containing metabolites revealed an accumulation of labelled ethanolamine when the heart was perfused with - 0.4 μM labelled ethanolamine. The changes in radioactivity distribution indicated that the phosphorylation of ethanolamine had become rate-limiting in the CDP-ethanolamine pathway when the heart was perfused with - 0.4 μM ethanolamine. Perfusion with different concentrations of ethanolamine did not significantly change the intracellular ethanolamine pool. The accumulation of labelled ethanolamine without a corresponding change in the ethanolamine pool suggests that the newly imported ethanolamine did not equilibrate with the endogenous ethanolamine pool. We postulate that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis.

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.

Synthesis of Ethanolamine and Its Regulation in Lemna paucicostata

The metabolism of ethanolamine and its derivatives in Lemna paucicostata has been investigated, with emphasis on the path-way for synthesis of phosphoethanolamine, a precursor of phosphatidylcholine in higher plants. In experiments involving labeling of intact plants with radioactive serine, ambiguities of interpretation due to entry of radioactivity into methyl groups of methylated ethanolamine derivatives were mitigated by pregrowth of plants with methionine. Difficulties due to labeling of diacylglyceryl moieties of phospholipids were avoided by acid hydrolysis of crucial samples and determination of radioactivity in isolated serine or ethanolamine moieties. The results obtained from such experiments are most readily reconciled with the biosynthetic sequence: serine --> ethanolamine --> phosphoethanolamine --> phosphatidylethanolamine. A possible alternative is: serine --> phosphatidylserine --> phosphatidylethanolamine --> ethanolamine --> phosphoethanolamine. Cell-free extracts of L. paucicostata were shown to produce CO(2) from the carbon originating as C-1 of serine at a rate sufficient to satisfy the demand for ethanolamine moieties. A number of experiments produced no support for a hypothetical role for phosphoserine in phosphoethanolamine formation. Uptake of exogenous ethanolamine commensurately down-regulates the synthesis of ethanolamine moieties (considered as a whole, and regardless of their state of derivatization at the time of their formation). In agreement with previous observations, uptake of exogenous choline down-regulates the methylation of phosphoethanolamine, without being accompanied by secondary accumulation of a marked excess of ethanolamine derivatives.

Effect of phosphatidylethanolamine and its constituent base on the metabolism of linoleic acid in rat liver

Dietary phosphatidylethanolamine (PE) contributes the circulatory and hepatic free-ethanolamine in rats (Ikeda et al. (1987) Biochim. Biophys. Acta 921, 245). A role for circulatory ethanolamine has not been defined; however, our recent studies have shown that exogenous ethanolamine influences cholesterol and linoleic acid metabolism in rats (Imaizumi et al. (1983) J. Nutr. 113, 2403). In order to understand the role of dietary PE the effects of PE and its base on the hepatic metabolism of linoleic acid were investigated in vivo and in primary cultured hepatocytes in rats. Dietary PE increased the plasmic level of ethanolamine from 37 to 52 μM and decreased the ratio of arachidonate to linoleate in hepatic phospholipids. Activity of hepaticΔ 6-desaturase decreased in rats given PE and the desaturation of [ 14C]linoleate in the cultured hepatocytes decreased by the addition of ethanolamine. Secretion of [ 14C]linoleate labeled very-low-density lipoprotein from the cultured hepatocytes decreased by the addition of ethanolamine. Dietary PE caused an increased formation of CO 2 from [ 14C]acetate by liver slices, and ethanolamine added to the hepatocytes caused an increased oxidation of [ 14C]linoleate and a suppression of fatty acid synthesis from [ 3H]serine. These results suggest that ethanolamine derived from the dietary PE plays a regulatory role in the linoleate metabolism in the liver.

The Levels of Phosphoethanolamine and Phosphocholine in Intact Neuroblastoma Cells: Effects of Cell Differentiation and of Exogenous Ethanolamine and Choline

Annals of the New York Academy of SciencesVolume 508, Issue 1 p. 474-475 The Levels of Phosphoethanolamine and Phosphocholine in Intact Neuroblastoma Cells: Effects of Cell Differentiation and of Exogenous Ethanolamine and Choline P. GLYNN, P. GLYNN A T& T Bell Laboratories 600 Mountain Avenue Murray Hill, New Jersey 07974Search for more papers by this authorT. M. LEE, T. M. LEE A T& T Bell Laboratories 600 Mountain Avenue Murray Hill, New Jersey 07974Search for more papers by this authorS. OGAWA, S. OGAWA A T& T Bell Laboratories 600 Mountain Avenue Murray Hill, New Jersey 07974Search for more papers by this author P. GLYNN, P. GLYNN A T& T Bell Laboratories 600 Mountain Avenue Murray Hill, New Jersey 07974Search for more papers by this authorT. M. LEE, T. M. LEE A T& T Bell Laboratories 600 Mountain Avenue Murray Hill, New Jersey 07974Search for more papers by this authorS. OGAWA, S. OGAWA A T& T Bell Laboratories 600 Mountain Avenue Murray Hill, New Jersey 07974Search for more papers by this author First published: November 1987 https://doi.org/10.1111/j.1749-6632.1987.tb32941.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume508, Issue1Physiological NMR Spectroscopy: From Isolated Cells to ManNovember 1987Pages 474-475 RelatedInformation