Evidence that the enzymes involved in the methylation of phosphatidylethanolamine are on the external side of the microsomal vesicles

The topology of the phosphatidylethanolamine (PE)-N-methyltransferase(s) on rat liver microsomes has been studied. The activity for the conversion of PE to phosphatidylcholine decreased by 50% after 2 min of exposure of the microsomes to trypsin and was virtually eliminated with 15 min. When exogenous monomethyl-PE or dimethyl-PE were incubated with microsomes, the formation of dimethyl-PE and phosphatidylcholine were also eliminated as a result of trypsin digestion. During the experiments the microsomes remained intact, since the latency of the mannose-6-phosphate phosphohydrolase remained approx. 90%. It is concluded that the active site(s) of the enzyme(s), or portions of the enzyme(s) indispensable to its activity, are present at the cytosolic side of the microsomes.     

Evidence that cholic acid CoA ligase is located asymmetrically on the cytoplasmic surface of hepatic microsomal vesicles.

The cholic acid CoA ligase activity of rat liver was quantitatively inactivated by proteolysis with pronase, chymotrypsin, subtilisin, or proteinase K in intact microsomal vesicles. Under the conditions employed, less than 14% of the lumenal mannose-6-phosphate phosphatase activity was lost, and the mannose-6-phosphate phosphatase activity remained highly latent. After microsomal integrity was disrupted with sodium deoxycholate, protease treatment resulted in a loss of greater than 74% of the mannose-6-phosphate phosphatase activity. Cholic acid CoA ligase activity was unaffected by preincubation of microsomes with sodium taurocholate under conditions that led to the complete expression of latent mannose-6-phosphate phosphatase activity. The data suggest that cholic acid CoA ligase activity is located on the cytoplasmic surface of hepatic microsomal vesicles.     

Topography of phosphatidylcholine,phosphatidylethanolamine and triacylglycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of these enzymes are located on the external surface of microsomal vesicles.It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Evidence that only newly made phosphatidylethanolamine is methylated to phosphatidylcholine and that phosphatidylethanolamine is not significantly deacylated-reacylated in rat hepatocytes

The metabolism of the molecular species of phosphatidylethanolamine derived from [3H]ethanolamine and molecular species of phosphatidylcholine derived from [3H]ethanolamine or [methyl-3H]choline has been studied in rat hepatocytes. After an initial pulse of radioactivity for 1 h and a chase for up to 24 h, the cells were harvested and the incorporation of label into the various molecular species of phosphatidylethanolamine and phosphatidylcholine was determined. The incorporation and metabolism of choline- and ethanolamine-labeled phosphatidylcholine was consistent with deacylation of some species of phosphatidylcholine and reacylation to form molecular species of phosphatidylcholine with different fatty acyl components. In contrast, such remodeling of ethanolamine-labeled phosphatidylethanolamine was not evident. Radioactivity disappeared from all molecular species of phosphatidylethanolamine without an increase in any of the species of phosphatidylethanolamine. This radioactivity was recovered in water-soluble metabolites in the cells and medium. Phosphatidylethanolamine (16:0-22:6) had an initial turnover rate (5.8 nmol/h) which was two or more times that of any of the other major molecular species of phosphatidylethanolamine. The molecular species of phosphatidylethanolamine displayed biphasic turnover profiles. The second rate of decay of radioactivity between 12 and 24 h was 2-4 times slower than the initial decay rate. During the first 2 h of the chase period, phosphatidylcholine was a major metabolite of labeled phosphatidylethanolamine. Subsequently, there was minimal conversion of phosphatidylethanolamine to phosphatidylcholine which suggests that only newly made phosphatidylethanolamine is available as a substrate for methylation to phosphatidylcholine.     

Headgroup biosynthesis of phosphatidylcholine and phosphatidylethanolamine in seed plants

Phospholipid biosynthesis is crucial for plant growth and development. It involves attachment of fatty acids to a phospho-diacylglycerol backbone and modification of the phospho-group into an amino alcohol. The biochemistry and molecular biology of the former has been well established, but a number of enzymes responsible for the latter have only recently been cloned and functionally characterized in Arabidopsis and some other model plant species. The metabolism involving the polar head groups of phospholipids established by past biochemical studies can now be validated by available gene knockout models. Moreover, gene knockout studies have revealed emerging functions of phospholipids in regulating plant growth and development. This review aims to revisit the old questions of polar headgroup biosynthesis of plant phosphatidylcholine and phosphatidylethanolamine by giving an overview of recent advances in the field and beyond.     

Co-ordinate control of CDP-choline and phosphatidylethanolamine methyltransferase pathways for phosphatidylcholine biosynthesis occurs in response to change in diet fat

Brain microsomal and synaptic plasma membrane phosphatidylcholine composition and biosynthetic activity were examined in relation to the composition of diet fat fed. Phosphocholinetransferase and methyltransferase activities are shown to be modulated by the diet, and by changes in the membrane phospholipid content of long-chain polyunsaturated fatty acids. This physiological modulation is co-ordinated such that the rate of phosphatidylcholine synthesis via one route is inversely regulated with activity of the alternate pathway.     

Topography of glycerolipid synthetic enzymes. Synthesis of phosphatidylserine, phosphatidylinositol and glycerolipid intermediates occurs on the cytoplasmic surface of rat liver microsomal vesicles

The topography of glycerolipid biosynthetic enzymes within the transverse plane of rat liver microsomal vesicles was investigated: (1) by use of the impermeant inhibitor, mercury-dextran; (2) by use of proteases; and (3) by determining whether the enzyme activities are latent. The seven enzyme activities investigated (dihydroxyacetone-phosphate acyltransferase, acyldihydroxyacetone-phosphate oxidoreductase, phosphatidic acid : CTPcytidyltransferase, CDPdiacylglycerol : inositol phosphatidyltransferase, 2-monoacylglycerol acyltransferase, diacylglycerol kinase, and the serine base exchange enzyme) function in phosphatidylinositol and phosphatidylserine synthesis and at intermediate levels in glycerolipid synthesis including steps of ether lipid synthesis. Mercury-dextran inhibited four of these enzymes greater than 60% in intact microsomal vesicles. One or more of the proteases employed (chymotrypsin, trypsin and pronase) inactivated each of the seven enzyme activities in intact microsomal vesicles. These two approaches indicate that each of these enzymes has important domains located on the cytoplasmic surface of microsomal vesicles. These enzyme activities could be assayed in intact microsomal vesicles. None appeared to be highly latent, indicating that substrates have free access to active sites. One substrate for each of these enzymes had been shown previously to be unable to cross the microsomal membrane. These data indicate that the active sites of these enzymes are located on the cytoplasmic surface of microsomal vesicles. It is concluded that the synthesis of phosphatidylserine and phosphatidylinositol, intermediates of ether lipid formation and other intermediates of glycerolipid synthesis occur asymmetrically on the cytoplasmic surface of the endoplasmic reticulum. These findings and our previous investigations on the topography of seven enzymes of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine biosynthesis (Ballas, L.M. and Bell, R.M., Biochim. Biophys. Acta 602, (1980) 578-590) indicate that the synthesis of the major cellular glycerolipids occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Transbilayer exchange of phosphatidylethanolamine for phosphatidylcholine and N-acetimidoylphosphatidylethanolamine in single-walled bilayer vesicles.

A preparation of small single-walled liposome vesicles containing a 9:1 mole ratio of phosphatidylcholine to phosphatidylethanolamine was subjected to reaction with the membrane-impermeable reagent, isethionyl acetimidate hydrochloride. This reagent converted 90% of the external phosphatidylethanolamine groups to the amidine derivative, leaving the mole ratio of unreacted phosphatidylethanolamine to phosphatidylcholine on the outside surface of the vesicle much lower than that on the inside surface. Equilibration of phosphatidylethanolamine across the bilayer was then measured as a function of time by monitoring the appearance of phosphatidylethanolamine on the outside surface utilizing the reaction of the amino groups with 2, 4, 6-trinitrobenzenesulfonic acid. The results show that no new phosphatidylethanolamine appeared on the external surface of the vesicles over a period of 12 days at 22 degrees. A conservative estimate of the precision of the measurements is +/- 10%. On this basis, the estimated half-time for the equilibration of phosphatidylethanolamine across the bilayer of these vesicles must be at least 80 days at 22 degrees.     

The effect of copper deficiency on heart microsomal phosphatidylcholine biosynthesis and concentration

The effect of dietary copper deficiency on phosphatidylcholine biosynthetic enzymes, phosphatidylethanolamine methyltransferase, phosphatidyldimethyltransferase and choline phosphotransferase of heart microsomes was measured in rats. The data indicated that dietary copper deficiency can alter phosphatidylcholine biosynthesis and concentration in microsomal membranes of the heart. There was a significant decrease in the specific activity of choline phosphotransferase. There was a significant decrease in the concentration of total phospholipid-P, phosphatidylcholine-P, phosphatidylethanolamine-P, phosphatidylinositol-P, sphingomyelin-P and cardiolipin-P in the microsomes of the copper deficient animals. There was a significant decrease in the concentration of copper in microsomes of heart and liver in the copper deficient animals.     

Kinetic analyses of liver phosphatidylcholine and phosphatidylethanolamine biosynthesis using 13C NMR spectroscopy

Choline and ethanolamine are substrates for de novo synthesis of phosphatidylcholine (PtdC) and phosphatidylethanolamine (PtdE) through the CDP-choline and CDP-ethanolamine pathways. In liver, PtdE can also be converted to PtdC by PtdE N-methyltransferase (PEMT). We investigated these kinetics in rat liver during a 60 min infusion with ¹³C-labeled choline and ethanolamine. NMR analyses of liver extracts provided concentrations and ¹³C enrichments of phosphocholine (Pcho), phosphoethanolamine (Peth), PtdC, and PtdE. Kinetic models showed that the de novo and PEMT pathways are ‘channeled’ processes. The intermediary metabolites directly derived from exogenous choline and ethanolamine do not completely mix with the intracellular pools, but are preferentially used for phospholipid synthesis. Of the newly synthesized PtdC, about 70% was derived de novo and 30% was by PEMT. PtdC and PtdE de novo syntheses displayed different kinetics. A simple model assuming constant fluxes yielded a modest fit to the data; allowing upregulated fluxes significantly improved the fit. The ethanolamine-to-Peth flux exceeded choline-to-Pcho, and the rate of PtdE synthesis (1.04 μmol/h/g liver) was 2–3 times greater than that of PtdC de novo synthesis. The metabolic pathway information provided by these studies makes the NMR method superior to earlier radioisotope studies.     

Structural differences among phosphatidylcholine, phosphatidylethanolamine, and mixed phosphatidylcholine/phosphatidylethanolamine multilayers: an infrared absorption study

We have examined the infrared absorption spectra from 4000 to 250 cm^?1 of multilayers of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylcholine/phosphatidylethanolamine (1:1 m/m) as a function of hydration, pH, and fatty acid composition. Characteristic splittings of the CH₂ bending and rocking modes and the position of the phosphoryl absorption at ca. 1240 cm^?1 reveal differences in acyl chain packing and head group conformation in the various films. Spectra demonstrate the importance of NH → O hydrogen bonding of the ethanolamine head group and the prerequisite head group conformation (tangent to the multilayer plane) in establishing these structural differences. The general appearance of the P-O-C stretching region (~1050 cm^?1) in the pure and mixed films further supports these conclusions and shows that the spectra clearly distinguish among the different head group orientations. Self-association of phosphatidylethanolamine is sometimes sufficient to prevent formation of mixed phases with phosphatidylcholine at neutral pH. The amount of fine structure, particularly in the low-frequency (800?200 cm^?1) region, in spectra of films of anhydrous, saturated-chain phospholipids decreases considerably when the films are monohydrated, when mixed phases exist, or when there are unsaturations in the acyl chains. These changes likely result from decreased crystal field effects in the spectra as the phosphatide packing density is decreased by any of the above procedures. Furthermore, the absence of other changes upon complete hydration of phosphatidylcholine films suggests that only the initial water is tightly bound to the lipid.     

Effect of choline deficiency on the enzymes that synthesize phosphatidylcholine and phosphatidylethanolamine in rat liver

Activities have been determined in subcellular fractions of livers from choline-deficient and normals rats for the enzymes that convert choline and ethanolamine to phosphatidylcholine and phosphatidylethanolamine respectively, that methylate phosphatidylethanolamine to yield phosphatidylcholine, and that oxidize choline to betaine. The activities of ethanolamine kinase, phosphoethanolamine cytidylyltransferase, and CDP-ethanolamine: 1,2-diacylglycerol phosphoethanolaminetransferase are not changed in the livers from choline-deficient rats for at least 18 days. Similarly, the activities of choline kinase and CDP-choline: 1,2-diacylglycerol phosphocholine transferase were unaffected by choline depletion. A decrease of 30-41% was observed, however, in the mitochondrial oxidation of choline to betaine. Also, the activity of the phosphocholine cytidylyltransferase was reduced in the choline-deficient livers to 60% olf the control values. The only observed increase in enzyme activity was a 62% elevation of the phosphatidylethanolamine-S-adenosylmethionine methyltransferase activity after 2 days of choline deficiency. This increased activity was maintained for at least 18 days of choline deprivation. The results suggest a lack of adaptive change in the levels of these phospholipid biosynthetic enzymes as a result of choline deficiency.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Evidence that phosphatidylcholine and phosphatidylethanolamine are synthesized by a single enzyme present in the endoplasmic reticulum of castor-bean endosperm.

Increasing concentrations of CDP-choline progressively inhibit the measured incorporation of CDP-[2-14C]ethanolamine into phosphatidylethanolamine catalysed by the ethanolaminephosphotransferase present in endoplasmic-reticulum membranes isolated from castor-bean endosperm cells. This inhibition parallels that observed during CDP-[Me-14C]choline incorporation and suggests that a single enzyme utilizes both these substrates.     

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides.

Phospholipids and sphingolipids are important precursors of lipid-derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross-talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat-2 fibroblasts. Addition of short-chain C6-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 microM C6-ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time-periods (>/= 16 h) to 50 microM C6-ceramide resulted in apoptosis. The structural analogue dihydro-C6-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-choline and CDP-ethanolamine under the influence of C6-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6-ceramide-treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6-ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase.     

Permeability of phosphatidylcholine and phosphatidylethanolamine bilayers

Sodium and glucose effluxes were measured in liposomes formed from a series of saturated phosphatidylcholines (PC) and phosphatidylethanolamines (PE). Vesicles composed of a saturated PC display a local permeability maximum in the region of the lipid transition temperature. The height of this maximum is predominantly a function of the thickness of the hydrocarbon chain region. Liposomes formed from a saturated PE do not display such a permeability maximum and in these vesicles the permeability process appears to be controlled by the head group region. It is postulated that the control exerted by the ethanolamine group is due to the reorganization of water structure it induces at the bilayer surface.     

Surface distribution of the fatty acid side chains of phosphatidylethanolamine in mixed phospholipid vesicles

A method has been developed for the selective determination of the fatty acid side chain distribution associated with the amino containing phospholipids located in the inner and outer surfaces of membranes. Using sonicated phosphatidylethanolamine/phosphatidylcholine vesicles as a model, the analysis consists of selective labeling of the outer surface amino groups with the membrane impermeable reagent 2,4,6-trinitrobenzenesulfonic acid. Outer and inner surface phosphatidylethanolamine fractions are separated by thin-layer chromatography. Analysis of methyl esters derived from these two fractions, by gas-liquid chromatography, yields the fatty acid side chain distribution. Our results show that there is no mol fraction dependence of the incorporation of any specific fatty acid side chains of egg yolk phosphatidylethanolamine into the vesicle or any preferential distribution of these side chains in the inner or outer vesicle surface. The surface distribution of the egg yolk phosphatidylethanolamine molecules in these vesicles appears to be determined by the head group packing requirements and not the fatty acid side chain composition.