Metabolism of the ethanolamine phosphoglycerides of mouse brain myelin and microsomes

Abstract— Three groups of six mice each were killed 1, 4 and 7 days after an intracerebral injection of [1,2-¹⁴C]ethanolamine. The specific radioactivities of the acid-labile ethanolamine phosphoglycerides (ethanolamine plasmalogens) and of the acid-stable ethanolamine phosphoglycerides (diacyl and alkyl acyl glycerophosphoryletholamines) from myelin and microsomal fractions were determined. All of these brain ethanolamine phosphoglycerides turn over rapidly with an apparent half-life of less than 3 days. The biosynthesis of alkenyl acyl glycerophosphorylethanolamines from diacyl glycerophosphorylethanolamines in mouse brain myelin or microsomes is unlikely.     

Enzymic synthesis of ether types of choline and ethanolamine phosphoglycerides by microsomal fractions from rat brain and liver.

The formation of product by ethanolamine phosphotransferases (EC 2.7.8.1) and cholinephosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats is increased several fold by 1,2-diacyl-sn-glycerols. With the addition of 1-alkyl-2-acyl-sn-glycerols, we have found an 11-fold increase with brain microsomes and a 20-fold increase with lvier microsomes in the synthesis of choline ether lipids (1-alkyl-2-acyl- and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylcholines). For the synthesis of ethanolamine ether lipids (1-alkyl-2-acyl and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines), the stimulation of alkylacylglycerols was 7-fold for brain microsomes and 18-fold for liver microsomes. The alkylacyl glycerols (8 mM) also inhibited the synthesis of diacyl phosphoglycerides by 44 to 65%, indicating that the same ethanolaminephosphotransferases and cholinephosphotransferases are utilized for the synthesis of alkylacyl phosphoglycerides and diacyl phosphoglycerides. A desaturation of the alkyl groups may take place in the same reaction mixture. The rate of incorporation of phosphorylcholine into alkenylacyl glycerophosphorylcholines (choline plasmalogens) with alkylacylglycerols, cytidine diphosphate choline, and liver microsomes was 15 nmoles per mg protein per hour. The in vitro synthesis of choline plasmalogens with alkylacylglycerols had not been observed previously. The corresponding rate of incorporation of phosphorylethanolamine into ethanolamine plasmalogens was 10 nmoles per mg protein per hour, a value greater than any of the previously reported values for ethanolamine plasmalogen formation from alkylacyl glycerophosphorylethanolamines.     

The synthesis of choline and ethanolamine phosphoglycerides in neuronal and glial cells of rabbit in vitro

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine-5′-phosphate choline (cytidine-5′-phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell-enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine-5′-phosphate choline and cytidine-5′-phosphate ethanolamine, respectively. Several kinetic properties of the choline-phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. K_m of cytidine-5′-phosphate ethanolamine, K_m of diacyl glycerol, pH optimum, need for divalent cations), but the K_m value for cytidine-5′-phosphate choline in glial cells was much lower (2.3 × 10^?4m ) than in neurons (1 × 10^?3m ). The K_mfor cytidine-5′-phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine-dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.     

Incorporation of [2-3H] ethanolamine into rat muscle phosphoglycerides

The contribution of phosphatidylethanolamine methylation to phosphatidylcholine biosynthesis in rat muscle was investigated by studying the incorporation of [2-3H] ethanolamine. The specific radioactivities of individual molecular species of muscle phosphoglycerides were measured by a combination of argentation thin-layer chromatography and countercurrent distribution. The specific radioactivity of phosphatidylethanolamine was approximately one thousand times that of phosphatidylcholine. Amongst individual phosphatidylethanolamines, hexaenoic species possessed the highest specific radioactivities and tetraenoic the lowest. Because of the very low incorporation into phosphatidylcholine, the specific radioactivities of combined rather than of individual fractions were measured. The results indicate that the contribution of phosphatidylethanolamine methylation to the overall biosynthesis of phosphatidylcholine in muscle is of minor importance.     

Effects of free fatty acids on the enzymic synthesis of diacyl and ether types of choline and ethanolamine phosphoglycerides.

Activities of ethanolaminephosphotransferases (EC 2.7.8.1) and choline phosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats are increased several fold by the addition of 1,2-diacyl-sn-glycerols or 1-alkyl-2-acyl-sn-clycerols. Oleic acid added with diacylglycerols stimulated further the synthesis of lecithins by liver microsomes, confirming the work of Sribney and Lyman (Can J. Biochem. 51: 1479-1486, 1973). With alkylacylglycerols, oleic and stearic acids were inhibitory and linoleic acid was even more inhibitory for the synthesis of both 1-alkyl-1-acyl-sn-glycero-3-phosphorylcholines and the corresponding ethanolamine compounds with microsomes from both tissues. Free fatty acids without added diglycerides had mixed effects. These results are best explained by postulating the presence of two isoenzymes each for ethanolaminephosphotransferase and cholinephosphotransferase of which only one is affected by free fatty acids. Regulation of the phosphotransferases by free fatty acids may determine the proportion of CDP-choline and CDP-ethanolamine used for synthesis of diacyl and alkylacyl types of these phosphoglycerides.     

Synthesis of ethanolamine phosphoglycerides by human platelets.

Platelet homogenates contain an ethanolaminephosphotransferase (EC 2.7.8.1) that catalyzes the synthesis of ethanolamine phosphoglycerides from cytidine-5'-diphosphate ethanolamine and 1-radyl-2-acyl-sn-glycerols. The enzyme is particulate-bound and requires Mn2+ and bile salts for optimal activity. The apparent Km of the enzyme for cytidine-5'-diphosphate ethanolamine is 1.6 X 10(-5) M when the concentration of 1,2-diacyl-sn-glycerols is 8.8 X 10(-4) M. The pH optimum is 8.5 in Tris-HCl or glycine-NaOH buffer. The activity of the enzyme in platelets from normal subjects is 0.24-0.34 nmole/min/mg of protein.     

Purification and characterization of choline/ethanolamine kinase from rat liver

Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa.     

Biosynthesis of phosphatidylethanolamines and phosphatidylcholines from ethanolamine and choline in rat liver.

1. The kinetics of phosphatidylcholine and phosphatidylethanolamine synthesis in rat liver were followed 5-60 min after the intraportal injection of [14-C]choline and [3-H]-ethanolamine. 2. At all time-intervals the specific radioactivity of CDP-choline was only about half that of phosphorylcholine. This indicated that CDP-choline was formed at a similar rate from phosphorylcholine and phosphatidylcholines, the latter probably through the reverse reaction of cholinephosphotransferase (EC 2.7.8.2.). In view of recent data obtained from experiments in vitro this implies a significant role for the cholinephosphotransferase reaction in the turnover of molecular species of phosphatidylcholine. 3. The specific radioactivity of CDP-ethanolamine was about twice that of phosphorylethanolamine at all time-intervals studied. This supports a previous suggestion that the liver phosphorylethanolamine pool is subject to compartmentation and shows that there is no rapid equilibration between different pools. In contrast with a recent study, no evidence was found for any significant methylation of phosphoryl-or CDP-ethanolamine to the corresponding choline derivative. 4. Quantitative data on the biosynthesis of molecular species of phosphoLIPIDS via CDP derivatives were calculated according to simple kinetic models. They were in the same range as those calculated from earlier data on precusors incorporated via diacylglycerols. 5. The proportion of radioactive phosphatidylethanolamines appearing in the plasma was approximately ten times lower than that for phosphatidylcholines. No selectivity was observed in the transfer into plasma of different molecular species of phosphatidylethanolamine.     

Effects of analogues of ethanolamine and choline on phospholipid metabolism in rat hepatocytes

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [(14)C]ethanolamine incorporation into phospholipids, whereas the incorporation of [(3)H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [(3)H]glycerol and hepatocytes, the appearance of (3)H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [(3)H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of (3)H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-(14)C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [(14)C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.     

Regulation of ethanolamine and choline phosphatide biosynthesis in isolated rat intestinal villus cells

The effects of ethanolamine, choline, and different fatty acids on phospholipid synthesis via the CDP-ester pathways were studied in isolated rat intestinal villus cells. The incorporation of [14C]glucose into phosphatidylethanolamine was stimulated severalfold by the addition of ethanolamine and long-chained unsaturated fatty acids, while the addition of lauric acid inhibited the incorporation of radioactivity into phosphatidylethanolamine. At concentrations of ethanolamine higher than 0.2 mM, phosphoethanolamine accumulated, but the concentrations of CDP-ethanolamine and the incorporation of radioactivity into phospatidylethanolamine did not increase further. The incorporation of [14C]glucose into phosphatidylcholine responded in a way similar to that of phosphatidylethanolamine, except that a 10-fold higher concentration of choline was required for maximal stimulation. CCC inhibited the incorporation of choline into phosphatidylcholine. In contrast with hepatocytes, villus cells did not form phosphatidylcholine via phospholipid N-methylation. The data indicate that, in intestinal villus cells, the cytidylyltransferase reactions are rate limiting in the synthesis of phosphatidylethanolamine and probably also of phosphatidylcholine. The availability of diacylglycerol and its fatty acid composition may also significantly affect the rate of phospholipid synthesis.     

Statistical analysis of the processes controlling choline and ethanolamine glycerophospholipid molecular species composition

The regulation and maintenance of the cellular lipidome through biosynthetic, remodeling, and catabolic mechanisms are critical for biological homeostasis during development, health and disease. These complex mechanisms control the architectures of lipid molecular species, which have diverse yet highly regulated fatty acid chains at both the sn1 and sn2 positions. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) serve as the predominant biophysical scaffolds in membranes, acting as reservoirs for potent lipid signals and regulating numerous enzymatic processes. Here we report the first rigorous computational dissection of the mechanisms influencing PC and PE molecular architectures from high-throughput shotgun lipidomic data. Using novel statistical approaches, we have analyzed multidimensional mass spectrometry-based shotgun lipidomic data from developmental mouse heart and mature mouse heart, lung, brain, and liver tissues. We show that in PC and PE, sn1 and sn2 positions are largely independent, though for low abundance species regulatory processes may interact with both the sn1 and sn2 chain simultaneously, leading to cooperative effects. Chains with similar biochemical properties appear to be remodeled similarly. We also see that sn2 positions are more regulated than sn1, and that PC exhibits stronger cooperative effects than PE. A key aspect of our work is a novel statistically rigorous approach to determine cooperativity based on a modified Fisher's exact test using Markov Chain Monte Carlo sampling. This computational approach provides a novel tool for developing mechanistic insight into lipidomic regulation.     

Influence of choline and ethanolamine administration on choline and ethanolamine phosphorylating activities of mouse liver and kidney.

The administration of ethanolamine to adult male mice resulted in a significant increase in ethanolamine kinase activity in liver and kidney. Similarly, choline administration resulted in a significant increase in choline kinase activity in liver and kidney. The administration of ethanolamine resulted in enhancement of choline kinase activity concomitantly with ethanolamine kinase activity in liver and kidney. The administration of choline, however, did not result in any significant increase in ethanolamine kinase activity in liver or kidney. Cycloheximide administration along with choline-ethanolamine prevented the increase in kinase activity in liver and kidney. The results obtained have been discussed in relation to the regulatory role of choline kinase and ethanolamine kinase by de novo synthesis in response to enhanced substrate concentration, the secondary nature of choline kinase induction on ethanolamine administration, and possible distinction between choline kinase and ethanolamine kinase.     

Metabolism of lysophosphatidyl ethanolamine and lysophosphatidyl choline by homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages

A comparison has been made between the conversion of (32)P-labeled lysophosphatidyl ethanolamine (LPE) and lysophosphatidyl choline (LPC) to their respective acylated and deacylated derivatives by homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages. Synthesis of PE by both homogenates and of PC by macrophage homogenates proceeded to about the same extent and is attributed to direct acylation of the lyso compounds. At higher LPC concentrations formation of PC by leukocytes is far greater than by macrophages. The mechanism of this enhanced synthesis of PC, which is brought out by higher substrate concentrations, is believed to be a transfer of the acyl group of one LPC molecule to another. Under optimal conditions macrophage homogenates deacylated LPE to a greater extent than LPC, while the reverse was true for leukocyte homogenates. Albumin inhibited deacylation of LPC and its conversion to PC by leukocytes, perhaps by binding the substrate (2 moles of LPC per mole of albumin). Other effects of albumin-stimulation of deacylation and acylation of LPE by macrophages, inhibition of deacylation and acylation of LPE by leukocytes-remain unexplained.     

Semisynthetic preparation of choline and ethanolamine plasmalogens

Choline and ethanolamine plasmalogens containing defined acyl chains are prepared by deacylation and reacylation of beef heart plasmalogens. During the reactions, the amino group of ethanolamine plasmalogens is protected by the trityl group. Deacylation is achieved by mild alkaline hydrolysis, and the lysoplasmalogens are reacylated with oleoylimidazolide in the presence of the methylsulfinylmethide anion. The protective group is removed from N-trityl ethanolamine plasmalogen by treatment with silicic acid in hexane. The choline and ethanolamine plasmalogens prepared by the procedures described are free of geometric, positional and steric isomers.     

Turnover of ethanolamine phosphoglycerides in different brain areas of adult and aged rats

The turnover of ethanolamine glycerophospholipids (EGP) has been determined in six different cerebral areas of 4-month and 22-month-old rats, by injecting [³H]glycerol together with [¹⁴C]ethanolamine into the lateral ventricle of the brain. The areas examined behave quite differently in respect to their utilization of the most simple precursors of phosphoglyceride biosynthesis. The incorporation of both glycerol and ethanolamine is already complete as early as 2–4 hours and then reutilization begins, at least for the so called fast pools of phosphoglycerides. The different slopes of the specific activity of the two precursors in EGP suggest a high degree of variance among catabolic rates in the different brain regions. In aged rats the utilization of the water-soluble precursors of EGP synthesis decreases in all brain areas and these data suggest that aging may have a different effect on the catabolic activities as well as phospholipid biosynthesis.