Phospholipid synthesis in isolated fat cells. Studies of microsomal diacylglycerol cholinephosphotransferase and diacylglycerol ethanolaminephosphotransferase activities.

Diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in microsomes from isolated rat fat cells. Assays based on the conversion of CDP-[14C]choline of CDP-[14C]ethanolamine to phosphatidylcholine or phosphatidylethanolamine utilized ethanol-dispersed diacylglycerols and 1 to 5 microng of protein. Cholinephosphotransferase and ethanolaminephosphotransferase activities had similar dependences on MgCl2 and pH, and were inhibited similarly by CaCl2, organic solvents, Triton X-100, Tween 20, and dithiothreitol. Ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid stimulated both activities similarly. With 1,2-dioleoyl-sn-glycerol, the cholinephosphotransferase activity had an apparent Km for CDP-choline of 23.9 micronM and a V max of 8.54 nmol/min/mg. CDP-ethanolamine and CDP were competitive inhibitors of the cholinephosphotransferase activity (apparent Kl values of 227 micronM and 360 micronM, respectively). With 1,2-dioleoyl-sn-glycerol, the ethanolaminephosphotransferase activity had an apparent Km of 18.3 micronM for CDP-ethanolamine and a V max of 1.14 nmol/min/mg. CDP-choline appeared to be a noncompetitive inhibitor of the ethanolaminephosphotransferase activity (apparent Kl of 1620 micronM). Inhibition of the ethanolaminephosphotransferase activity by CDP appeared to be of a mixed type. The dependences on diacylglycerols containing fatty acids 6 to 18 carbons in length were investigated...     

Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn-glycerols.

The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 micron) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 micron was found to inhibit the rates of phophatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.     

Synthesis of phosphatidylcholine and phosphatidylethanolamine in relation to the concentration of membrane-bound diacylglycerols of rat lung microsomes

Different concentrations of membrane-bound diacylglycerol were generated in vitro in rat lung microsomes by treatment with CMP. Diacylglycerol concentrations of between 16 (endogenous content) and 48 nmol/mg of microsomal protein were obtained. The relative proportion of the disaturated species of diacylglycerol remained constant at all diacylglycerol concentrations. Choline- and ethanolaminephosphotransferase activity was determined in relation to the diacylglycerol concentrations of microsomes. The activity of both phosphotransferases increased. The relative proportion of disaturated phosphatidylcholine synthesized at each diacylglycerol concentration was nearly the same and corresponded to the relative proportion of the disaturated species in the diacylglycerol. Disaturated phosphatidylethanolamine was not formed. The affinities of the choline- and ethanolaminephosphotransferases for the diacylglycerol substrate were different. We conclude that the cholinephosphotransferase is generally non-selective for the diacylglycerol substrate. The available diacylglycerol pattern seems to govern the species pattern of phosphatidylcholine and phosphatidylethanolamine. The kinetics of the phosphotransferases regulate the mass proportion of these phospholipids.     

Topography of phosphatidylcholine, phosphatidylethanolamine and triacylgycerol 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), sn-glycerol-3-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 sn-glycerol-3-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 thease 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.     

Inhibition of phosphatidylethanolamine biosynthesis in baby hamster kidney-21 cells infected with Semliki Forest virus.

Semliki Forest virus inhibits phosphatidylethanolamine biosynthesis in baby hamster kidney-21 cells 6 h after infection. Viral infection reduced the incorporation of [1,2-14C]-ethanolamine into intact cells by approximately 50%. A similar reduction in the activity of the ethanolaminephosphotransferase (EC 2.7.8.1) was also observed. The apparent Km for CDPethanolamine was 60 muM for the microsomal enzymes from infected or mock-infected cells. In addition, exogenous diglyceride only stimulated by 1.5-fold the ethanolaminephosphotransferase from virus- or mock-infected cells, whereas the same diglyceride preparations stimulated the cholinephosphotransferase (EC 2.7.8.2) from baby hamster kidney cells by sixfold. Generation of endogenous diglyceride by pretreatment of the microsomes with phospholipase C (EC 3.1.4.3) stimulated the activity of the cholinephosphotransferase but not the ethanolaminephosphotranferase. Semliki Forest virus does not inhibit all microsomal enzymes, since the activities of NADH- K3Fe(CN)6 reductase and NADH dehydrogenase (EC 1.6.99.3) were not affected. The ethanolaminephosphotransferase from virus- and mock-infected cells showed similar profiles of activity as a function of temperature; this result and other studies suggest that that membranous environment of the ethanolaminephosphotransferase was not significantly modified by the virus.     

Effect of 1-oleoyl-2-acetylglycerol and other lipids on phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in cultured type II pneumocytes

We previously reported that addition of phosphatidylglycerol to the culture medium stimulates phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in type II pneumocytes. In view of the known biological effects of diacylglycerols and since phosphatidylglycerol could be metabolized to diacylglycerol, we now examined the effects of diacylglycerols on the same parameters. The rate of choline incorporation into phosphatidylcholine was increased 30-60% by 10 microM phosphatidylglycerol, diolein, mixed diacylglycerols and 1-oleoyl-2-acetylglycerol (OAG). The effects of phosphatidylglycerol and OAG were not additive, suggesting a similar mechanism of action. The diacylglycerols and phosphatidylglycerol increased the activity of cholinephosphate cytidylyltransferase in type II cell sonicates by 35-50%, but had no effect on the activities of choline kinase, cholinephosphotransferase or 1-acylglycerophosphocholine acyltransferase. Again, the effects of OAG and phosphatidylglycerol on cytidylyltransferase were not additive. It is known that addition of lipids to the assay mixture increases the activity of cholinephosphate cytidylyltransferase in vitro and inclusion of the above lipids (1.1 mM) in the in vitro assay mixture increased cytidylyltransferase activity in type II cell sonicates. In addition, the stimulatory effects of OAG and of diolein, as well as of phosphatidylglycerol as reported previously, in the culture medium on cytidylyltransferase activity in type II cells were diminished or abolished when the assay was carried out in the presence of sufficient amounts of the same lipids to stimulate maximally the activity in vitro. These data show that lipids in the culture medium stimulate phosphatidylcholine biosynthesis in type II cells by direct activation of cholinephosphate cytidylyltransferase.     

Cloning, genomic organization, and characterization of a human cholinephosphotransferase

A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated using in vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeast CPT1 gene in its interaction with SEC14 and affect cell growth.     

Utilization of endogenous diacylglycerol for the synthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine by lipid particles from baker's yeast (Saccharomyces cerevisiae).

The activity of the enzymes diacylglycerol acyltransferase (EC 2.3.1.20), cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) have been measured in a lipid particle preparation from baker's yeast (Saccharomyces cerevisiae) with endogenous 1,2-diacylglycerol as substrate. For all three enzymes the rate of diacylglycerol utilization was established with respect to substrate and Mg2+ concentration. Neither of the enzyme activities was stimulated significantly by addition of diacylglycerols. The conversion of diacylglycerol into triacylglycerol in the presence of CDP-choline and CDPethanolamine, and the synthesis of phospholipids in the presence of acyl-CoA either added or generated in situ were studied. Neither CDPcholine nor CDPethanolamine had an effect on triacylglycerol synthesis. Exogenous acyl-CoA had no effect on either choline- or ethanolaminephosphotransferase activity. However, when the necessary substrates for formation of acyl-CoAs in situ (ATP, CoA, Mg2+ and free fatty acids) were added a decrease in both cholinephosphotransferase and ethanolaminephosphotransferase activity was observed. This inhibition was shown to be due to ATP and might explained as a result of chelation of the Mg2+, a necessary activator of both the choline- and the ethanolaminephosphotransferase.     

Reversibility of the reactions catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase solubilized from rat-brain microsomes.

The incorporation of CMP into CDP-ethanolamine and CDP-choline, catalyzed by ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2), respectively, has been studied in solubilized preparations of rat-brain microsomes. Mn2+ ions were required for the maximal activity of both enzymes. The CMP concentration needed to reach the half-maximal reaction rate was 1.6 microM for both activities. The rate of incorporation of CMP into CDP-choline and CDP-ethanolamine was increased by increasing the concentration of phosphatidylcholine and phosphatidylethanolamine, respectively, in detergent-phospholipid micellar systems. The rate of the reaction at pH 6.5 was comparable with that measured at pH 8.5, whereas the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine, catalyzed by the same enzymes, increased with pH. Ethanolaminephosphotransferase, which catalyzes the synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol, was co-eluted with the enzyme activity catalyzing the reverse reaction, when solubilized microsomes were submitted to anion exchange chromatography on DEAE Bio-Gel A. Cholinephosphotransferase was inactivated during the chromatographic procedure.     

A study of the molecular species of diacylglycerol, phosphatidylcholine and phosphatidylethanolamine and of cholinephosphotransferase and ethanolaminephosphotransferase activities in the type II pneumocyte

The percent distributions of the molecular species of diacylglycerol, phosphatidylcholine and phosphatidylethanolamine in rat whole lung and type II pneumocytes were found to differ significantly. Diacylglycerol from the type II pneumocyte is enriched in the disaturated species and diminished in the polyenoic species compared to whole lung. Type II pneumocyte phosphatidylcholine is enriched in the disaturated species and diminished in all other species compared to whole lung. Relative to whole lung, type II pneumocyte phosphatidylethanolamine is greatly enriched in monoenoic and depleted in polyenoic fatty acid species. Analysis of the fatty acid composition of the molecular species in general indicated differences in relative amounts of fatty acids which were most pronounced in palmitic, palmitoleic, stearic and oleic acids, both within and between type II pneumocyte and whole lung glycerolipids. Significant differences between molecular species also existed within type II pneumocyte glycerolipids. In this cell type, phosphatidylcholine is enriched in disaturated and diminished in monoenoic species compared to diacylglycerol. Phosphatidylethanolamine is enriched in monoenoic and polyenoic species relative to diacylglycerol. In order to determine whether differences observed in type II pneumocyte glycerolipid molecular species were attributable to differences in the specificities of cholinephosphotransferase and ethanolaminephosphotransferase, the selectivity of these enzymes was examined. While cholinephosphotransferase showed diminished activity towards 1-stearoyl-2-linoleoyl-sn-glycerol, neither enzyme showed selectivity towards other tested diacylglycerols under a variety of conditions. Therefore, while in the type II pneumocyte significant amounts of phosphatidylcholine (particularly the disaturated species) and phosphatidylethanolamine may be synthesized de novo, enzymes responsible for remodeling (phospholipase A2 and acyltransferases) may play an important role in establishing the final molecular species composition of both phosphatidylcholine and phosphatidylethanolamine.     

Hamster liver cholinephosphotransferase and ethanolaminephosphotransferase are separate enzymes

CDP-choline:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) are microsomal enzymes that catalyze the final steps in the syntheses of phosphatidylcholine and phosphatidylethanolamine via the CDP-choline and CDP-ethanolamine pathways, respectively. Both enzyme activities were cosolubilized from hamster liver microsomes by Triton QS-15. Limited separation of these two activities was achieved by ion-exchange chromatography. The partially purified phosphotransferases displayed a higher sensitivity than microsomal phosphotransferases towards exogenous phospholipids and showed an absolute requirement for divalent cations. Upon purification, cholinephosphotransferase was more stable to heat treatment than ethanolaminephosphotransferase. The two enzymes exhibited distinct pH optima and responded differently to exogenous phospholipids. Our results clearly indicate that cholinephosphotransferase and ethanolaminephosphotransferase are separate enzymes.     

Phosphatidylcholine homeostasis in phosphatidylethanolamine-depleted Tetrahymena

The relative contributions of the two pathways of phosphatidylcholine biosynthesis, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17) and diacylglycerol: CDP-choline cholinephosphotransferase (EC 2.7.8.1), are altered in the ciliate protozoan Tetrahymena thermophila whose phospholipid composition has been modified by culturing the organism in the presence of one of several aminophosphonic acids, as determined by measuring the incorporation of [methyl-3H]choline and [methyl-14C]methionine into phosphatidylcholine in vivo. In control cells the phosphotransferase pathway provides about 40% of the phosphatidylcholine, while in cells grown with 2-aminoethylphosphonate (AEP), 3-aminopropylphosphonate (APP), and N,N,N-trimethylaminoethyl-phosphonate (TMAEP) the contribution of the phosphotransferase pathway to phosphatidylcholine formation is 75, 90, and 26%, respectively. In AEP- and APP-grown cells, in which 80% of the phosphatidylethanolamine has been replaced by the corresponding phosphonolipid, the methyltransferase is less active since the level of the substrate phosphatidylethanolamine is reduced and neither of the phosphonolipids is a substrate for the enzyme. In TMAEP-grown cells, TMAEP competes with and reduces the incorporation of phosphocholine by the phosphotransferase pathway, leading to a smaller contribution of the pathway to phosphatidylcholine biosynthesis. The relative amounts of the two different radioactive labels incorporated into diacylphosphatidylcholine vs alkylacylphosphatidylcholine are also altered in the phosphonate-grown cells. The exogenous AEP induces a change in the glyceryl ether content of the 2-aminoethylphosphonolipid--33% in the AEP-grown cells compared to 70% in the control cells--indicating that the exogenous AEP is entering the phospholipids by the ethanolamine-phosphotransferase pathway rather than by the route of the endogenous AEP.     

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.     

Effect of dimethylaminoethylphosphonate on phospholipid metabolism in Tetrahymena

Dimethylaminoethylphosphonate (DMAEP) was incorporated into the phospholipids of the ciliate protozoan Tetrahymena thermophila at the expense of both phosphatidylethanolamine and phosphatidylcholine, but it had no effect on the levels of the 2-aminoethylphosphonolipid. The newly formed DMAEP-lipid accounted for almost 50% of the phospholipids of the organism. The DMAEP was incorporated into the phospholipids using both the ethanolaminephosphotransferase and cholinephosphotransferase pathways. The DMAEP-lipid was not methylated to the trimethyl derivative, confirming the lack of methylation of phosphonolipids by Tetrahymena.     

Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers

CDPcholine: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in Plasmodium knowlesi-infected erythrocytes obtained from Macaca fascicularis monkeys. Disrupted infected erythrocytes possess a cholinephosphotransferase activity (1.3 +/- 0.2 nmol phosphatidylcholine/10(7) infected cells per h) 1.5-times higher than the ethanolaminephosphotransferase activity. Optimal activities of both enzymes were observed in the presence of 12 mM MnCl2, which was about 3-times as effective as 40 mM MgCl2 as a cofactor. The two activities had similar dependences on pH and thermal inactivation. Their Arrhenius plots show an identical break at 17 degrees C and the corresponding activation energies below and above the critical temperature were similar for the two activities. Sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, beta-D-octylglucoside and lysophosphatidylcholine strongly inhibited the two activities above their critical micellar concentration, but the first three detergents stimulated the activities at lower concentrations. Saponin (0.004-0.5%) either did not affect the two activities or else increased them. Cholinephosphotransferase and ethanolaminephosphotransferase activities had apparent Km values for the CDP ester of 23.4 and 18.6 microM, respectively. CDPcholine and CDPethanolamine competitively inhibited the ethanolaminephosphotransferase and cholinephosphotransferase activities, respectively. The high selectivity of these activities for individual molecular species of diradylglycerol suggests that substrate specificity is responsible for the various molecular species of Plasmodium-infected erythrocyte phospholipids. However, cholinephosphotransferase and ethanolaminephosphotransferase had different dependences on 1,2-dilauroylglycerol and 1-oleylglycerol, which were substrates for cholinephosphotransferase but not for ethanolaminephosphotransferase under our conditions. These data provide the first characterization of an enzyme involved in the intense lipid metabolism in Plasmodium-infected erythrocytes, and the presence of cholinephosphotransferase demonstrates a biosynthesis of phosphatidylcholine by the Kennedy pathway after infection. Our data suggest that cholinephosphotransferase and ethanolaminephosphotransferase activities could be catalyzed by the same enzyme. Furthermore, since host erythrocytes are devoid of these enzymatic activities, cholinephosphotransferase is a parasite-specific membrane-associated enzyme which can be used as a probe or marker.     

Purification of ethanolaminephosphotransferase from bovine liver microsomes

CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) has been purified to electrophoretic homogeneity and in a catalytically active form from bovine liver microsomes. The purification method is based on the high hydrophobicity of the protein whose charged sites appear to be masked from the interaction with the chromatographic stationary phases when membranes are solubilized with an excess of non-ionic detergent.The isolated protein has a molecular mass of about 38 kDa, as estimated by SDS-PAGE mobility, and exhibits both ethanolaminephosphotransferase and cholinephosphotransferase activities. Evidence is given that both activities are Mn²⁺-dependent and that the same catalytic site is involved in cholinephosphotransferase and ethanolaminephosphotransferase reactions. Mg²⁺-dependent CDP-choline:diacylglycerol cholinephosphotransferase (EC 2.7.8.2) is completely inactivated during the solubilization and purification steps.     

Studies of diacylglycerol cholinephosphotransferase and diacylglycerol ethanolaminephosphotransferase activities in Tetrahymena microsomes

Microsomes isolated from Tetrahymena pyriformis synthesized phosphatidylcholine and phosphatidylethanolamine by CDPcholine: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1), utilizing ethanol-dispersed dioleoglycerol. Cholinephosphotransferase and ethanolaminephosphotransferase activities have similar dependences on MgCl2 and MnCl2, but the latter was more effective than the former for both enzyme activities. The V values for 1,2-dioleoylglycerol obtained at optimal conditions were 1.8 nmol/min per mg microsomal protein for cholinephosphotransferase and 0.6 nmol/min per mg microsomal protein for ethanolaminephosphotransferase. Both enzymes could not utilize 1,3-dioleoylglycerol or 1-oleoylglycerol as substrates. Cholinephosphotransferase had an apparent Km for CDPcholine of 11.7 microM with 1,2-dioleoylglycerol and was inhibited by CDPethanolamine competitively. On the other hand, ethanolaminephosphotransferase has an apparent Km for CDPethanolamine of 8 microM and CDPcholine was a noncompetitive inhibitor of ethanolaminephosphotransferase activity. Furthermore, despite the marked alteration of phospholipid composition occurring during the temperature acclimation of Tetrahymena cells, both enzyme activities showed similar dependences on growth and incubation temperatures. This may imply that the final step of de novo synthesis of two major phospholipids does not participate in the thermally induced modification of the profile of phospholipid polar head group in membranes.     

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.     

Cholinephosphotransferase in rat lung. In vitro formation of dipalmitoylphosphatidylcholine and general lack of selectivity using endogenously generated diacylglycerol

Diacylglycerol was generated in vitro in rat lung microsomes by forming phosphatidic acid via sn-glycerol-3-phosphate acyltransferase followed by the hydrolysis of the phosphatidic acid by phosphatidate phosphohydrolase. Diacylglycerol concentrations of 35 to 50 nmol/mg of microsomal protein were obtained. Cholinephosphotransferase activity was determined in microsomes by measuring the conversion of endogenously generated [14C]diacylglycerol to phosphatidylcholine. Reaction rates of 14 to 16 nmol/min/mg of protein were obtained with a 30-s reaction. Diacylglycerol which was primarily dipalmitoylglycerol was produced when palmitic acid was used in the sn-glycerol-3-phosphate acyltransferase reactions. Dipalmitoylphosphatidylcholine was formed via cholinephosphotransferase from the dipalmitoylglycerol with an apparent maximal velocity of 20 nmol/min/mg of protein. When oleic acid was used instead of palmitic acid, the apparent maximal velocity for cholinephosphotransferase was 26 nmol/min/mg of protein. The apparent Km values for the two different diacylglycerol substrates were the same (28.5 nmol/mg of protein). Diacylglycerols, with different molecular species composition, were generated using a variety of fatty acids and fatty acid mixtures. The phosphatidylcholine formed from these diacylglycerols had the same molecular species profiles as the diacylglycerol used as the substrate. The relative reaction rates with the different diacylglycerols were essentially the same except when 20:4 and 22:6 fatty acids were used individually, in which case the rates were lower. We conclude that cholinephosphotransferase readily forms dipalmitoylphosphatidylcholine from endogenously generated dipalmitoylglycerol and that the cholinephosphotransferase reaction is generally nonselective for the diacylglycerol substrate.     

Phosphatidylcholine synthesis influences the diacylglycerol homeostasis required for SEC14p-dependent Golgi function and cell growth

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20-40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.