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.     

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.     

Preferential synthesis of diacyl and alkenylacyl ethanolamine and choline glycerophospholipids in rabbit platelet membranes

In rabbit platelet membranes, the contents of alkenylacyl phospholipids (plasmalogen) were 56% of phosphatidylethanolamine and 3% of phosphatidylcholine. This uneven distribution of plasmalogens in each phospholipid class could be attributed to the different substrate specificity of ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2). The properties of the enzymes were studied, using endogenous diglycerides and CDP-[3H]ethanolamine or CDP-[14C]choline as substrates. The newly formed phospholipids were mainly diacyl and alkenylacyl and only rarely alkylacyl type. The ratios of the labeled alkenylacyl to diacyl type of phospholipids clearly varied with the concentrations of CDP-ethanolamine or CDP-choline. When 1, 10, and 30 microM CDP-[3H]ethanolamine were used, the labeled phospholipids contained 53, 37, and 27% of the alkenylacyl type, respectively. The apparent Km for CDP-ethanolamine to synthesize alkenylacyl and diacyl types were 2.2 and 8.1 microM. On the other hand, when 1, 10, and 30 microM CDP-[14C]choline were used, the labeled lipids contained 10, 17, and 24% alkenylacyl type, respectively. The apparent Km for CDP-choline to synthesize alkenylacyl and diacyl types were 24 and 4.3 microM. Further, the syntheses of diacyl type of phosphatidylethanolamine and the alkenylacyl type of phosphatidylcholine were markedly inhibited by unlabeled CDP-choline and CDP-ethanolamine, respectively. The two enzymes had opposite substrate specificities, and ethanolaminephosphotransferase showed a high preference to plasmalogen synthesis, especially in the presence of CDP-choline.     

Phosphatidylethanolamine synthesis in castor bean endosperm

Phosphatidylethanolamine synthesis by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) from the endoplasmic reticulum of castor bean (Ricinus communis L. var. Hale) endosperm was characterized. The Michaelis-Menten constant of the enzyme for CDP-ethanolamine was approximately 8.0 micromolar. The pH optimum was 6.5 and a divalent cation was an absolute requirement for activity, with Mg²⁺ giving the greatest stimulation at 3 millimolar. Sulfhydryl reagents variously affected enzyme activity. No discernible differences were detected between the responses of the ethanolaminephosphotransferase and CDP-choline:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) to a variety of treatments. CDP-choline and CDP-ethanolamine were competitive inhibitors of the ethanolaminephosphotransferase and cholinephosphotransferase reactions, respectively.     

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.     

Ethanolaminephosphotransferase in rat lung: selectivity for endogenous and exogenous diacylglycerol

Ethanolaminephosphotransferase (EC 2.7.8.1) activity was determined in lung microsomes using diacylglycerols generated endogenously from [14C]glycerol 3-phosphate and different mixtures of fatty acids. Ethanolaminephosphotransferase used endogenously generated dipalmitoylglycerol better than dioleoylglycerol. The apparent Km and the reaction rates for four different endogenously generated mixtures were the same (16 nmol/mg microsomal proteins). The apparent Km values for CDP-ethanolamine were the same (0.26 mm) for endogenously generated dipalmitoylglycerol and dioleoylglycerol. The amount of diacylglycerol generated in microsomes was 2-3-times the apparent Km for diacylglycerol. Dipalmitoylglycerol, supplied exogenously as a Tween 20/phosphatidylglycerol emulsion, was nearly twice as active as dioleoylglycerol. Both dipalmitoylglycerol and dioleoylglycerol were more active as substrates when emulsions were made with phosphatidylglycerol/Tween 20 than with Tween 20 alone. The results suggest that ethanolaminephosphotransferase in lung is relatively nonselective for molecular species of diacylglycerol. In addition, the results suggest that the concentration of diacylglycerol and the physical state in which it is presented to the enzyme can affect the apparent selectivity of ethanolaminephosphotransferase for diacylglycerols.     

Solubilization and purification of rat liver microsomal 1,2-diacylglycerol: CDP-choline cholinephosphotransferase and 1,2-diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase.

1. The procedure, which involved 2-step sonication of microsomes at pH 7.4 and then at pH 8.5 in the presence of sodium deoxycholate and subsequent dialysis, resulted in 4-5-fold purification of choline-phosphotransferase and ethanolaminephosphotransferase with the yield of 40-50%. 2. Ethanolaminephosphotransferase was further purified 8.5-fold over microsomes by sucrose density gradient centrifugation of the partially purified preparation, while cholinephosphotransferase activity was considerably lost during this procedure. No separation of the two transferases from each other was achieved at this step. 3. Cholinephosphotransferase required Mg2+ as cofactor, and microsomal phospholipids for its maximal activity. On the other hand, Mn2+ was more effective than Mg2+ as cofactor for ethanol aminephosphotransferase, and this enzyme was inhibited by microsomal phospholipids. 4. Both transferases were stimulated several-fold by sodium deoxycholate and also showed similar optimal pH ranging from pH 8.0 to 8.5. 5. Km values for 1,2-diacylglycerol emulsion were 81.0 muM for cholinephosphotransferase and 63.0 muM for ethanolaminephosphotransferase, respectively. CDP-choline and CDP-ethanolamine competitively inhibited, with the same Ki value (both 350 muM), ethanolaminephosphotransferase and cholinephosphotransferase, respectively. The Ki values obtained were much greater than the corresponding Km values for the cytidine substrates (36.4 muM for CDP-choline and 22.0 muM for CDP-ethanolamine). 6. The partially purified enzymes were further treated with Triton X-100. When enzyme activities were assayed with Mg2+, cholinephosphotransferase, although considerably inactivated, was partially separated from ethanolaminephosphotransferase by sucrose density gradient centrifugation of Triton-treated preparations. Furthermore, cholinephosphotransferase (but not ethanol-aminephosphotransferase) itself was partially separated into Mg2+ -requiring and Mn2+ -requiring components. In contrast, ethanolaminephosphotransferase assayed with either Mg2+ or Mn2+ formed a single peak together with Mn2+ -requiring cholinephosphotransferase.     

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.     

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.     

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.     

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.     

Membrane lipid biosynthesis in Chlamydomonas reinhardtii: ethanolaminephosphotransferase is capable of synthesizing both phosphatidylcholine and phosphatidylethanolamine

Phosphatidylethanolamine, but not phosphatidylcholine, is found in Chlamydomonas reinhardtii. A cDNA coding for diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase (EPT) was cloned from C. reinhardtii. The C. reinhardtii EPT appears phylogenetically more similar to mammalian aminoalcoholphosphotransferases than to those of yeast and the least close to those of plants. Similar membrane topography was found between the C. reinhardtii EPT and the aminoalcoholphosphotransferases from mammals, yeast, and plants. A yeast mutant deficient in both cholinephosphotransferase and ethanolaminephosphotransferase was complemented by the C. reinhardtii EPT gene. Enzymatic assays of C. reinhardtii EPT from the complemented yeast microsomes demonstrated that the C. reinhardtii EPT synthesized both PC and PE in the transformed yeast. The addition of either unlabeled CDP-ethanolamine or CDP-choline to reactions reduced incorporation of radiolabeled CDP-choline and radiolabeled CDP-ethanolamine into phosphatidylcholine and phosphatidylethanolamine. EPT activity from the transformed yeast or C. reinhardtii cells was inhibited nearly identically by unlabeled CDP-choline, CDP-ethanolamine, and CMP when [14C]CDP-choline was used as the primary substrate, but differentially by unlabeled CDP-choline and CDP-ethanolamine when [14C]CDP-ethanolamine was the primary substrate. The Km value of the enzyme for CDP-choline was smaller than that for CDP-ethanolamine. This provides evidence that C. reinhardtii EPT, similar to plant aminoalcoholphosphotransferase, is capable of catalyzing the final step of phosphatidylcholine biosynthesis, as well as that of phosphatidylethanolamine in the Kennedy pathway.     

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.     

Isolation and characterization of a yeast mutant defective in cholinephosphotransferase

A yeast mutant defective in cholinephosphotransferase (cpt) was isolated as a revertant from a choline-sensitive mutant, which exhibited lowered phosphatidylinositol synthesis. A block at the cholinephosphotransferase step in the mutant was indicated by the enzyme defect and the accumulation of CDP-choline in the cells with a decrease in phosphatidylcholine synthesis. The defect was due to a single recessive mutation in a nuclear gene. The residual activity in the mutant showed an increased apparent Km for CDP-choline and an altered sensitivity to Tween 20. Thus the structural gene may be affected in the mutant. The occurrence of an intact ethanolaminephosphotransferase in the mutant indicates the distinctness of the genes encoding cholinephosphotransferase and ethanolaminephosphotransferase in yeast. The present selection method was also effective for isolating mutants defective in the other steps of the CDP-choline pathway and choline transport.     

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.     

Differential selectivity of cholinephosphotransferase and ethanolaminephosphotransferase of Tetrahymena for diacylglycerol and alkylacylglycerol

The glycerophospholipids of the ciliate protozoan Tetrahymena thermophila differ greatly in their content of alkylacylglycerol with phosphatidylcholine, phosphatidylethanolamine, and 2-aminoethylphosphonolipid containing 60, 4, and 53% glyceryl ether, respectively. This difference is achieved by differences in the selectivities of cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) for alkylacylglycerol and diacylglycerol. When the two enzymes are assayed in vitro using only endogenous diglyceride as substrate, the newly formed phosphatidylcholine contains 37% glyceryl ether, while the newly formed phosphatidylethanolamine contains 5% glyceryl ether. The ethanolaminephosphotransferase is stimulated equally well by addition of diolein and dipalmitin, but the diacylglycerols have no effect on the glyceryl ether content of phosphatidylethanolamine. In contrast, the glyceryl ether content of newly formed phosphatidylcholine decreases to 16% when the cholinephosphotransferase is exposed to diolein or dipalmitin. The ethanolaminephosphotransferase is not stimulated by addition of a 60:40 mixture of alkylacylglycerol/diacylglycerol. The cholinephosphotransferase is stimulated by the mixture to the same extent as it is by the diacylglycerols, with the glyceryl ether content of the newly formed phosphatidylcholine increasing to 52%. With the addition of alkylacylglycerol alone, the glyceryl ether content of the newly formed phosphatidylethanolamine increases to 10%, while that of the newly formed phosphatidylcholine increases almost to 60%.     

Mutants of Saccharomyces cerevisiae defective in sn-1,2-diacylglycerol cholinephosphotransferase. Isolation, characterization, and cloning of the CPT1 gene

A colony autoradiographic assay for the sn-1,2-diacylglycerol cholinephosphotransferase activity in Saccharomyces cerevisiae was developed. Twenty-two mutants defective in cholinephosphotransferase activity were isolated. Genetic analysis revealed that all of these mutations were recessive, and three complementation groups were identified. The cholinephosphotransferase activities in membranes prepared from cpt1 mutants were reduced 2-10-fold compared to wild-type activity. The cholinephosphotransferase activities of two cpt1 isolates differed from wild-type activity with respect to their apparent KM for CDP-choline. The residual cholinephosphotransferase activities of cpt1 isolates were more sensitive to inhibition by CMP than the wild-type activity. The CPT1 gene was cloned by genetic complementation of cpt1 using a yeast genomic library. In strains transformed with the CPT1-bearing plasmid, a 5-fold overproduction of cholinephosphotransferase activity with wild-type kinetic properties was observed. The CPT1 gene was localized to a 1.2-2.4-kilobase region of DNA by transposon Tn5 mutagenesis and deletion mapping. An insertional mutant of the CPT1 gene was constructed and introduced into the chromosome by integrative transformation. The resulting cpt insertional mutant fell into the cpt1 complementation group. The cholinephosphotransferase activity in membranes prepared from the cpt1 insertional mutant was reduced 5-fold and exhibited CMP sensitivity. The sn-1,2-diacylglycerol ethanolaminephosphotransferase activities in membranes from all of the cpt1 isolates including the insertional mutant were normal. The data indicate that the cloned CPT1 gene represents the yeast cholinephosphotransferase structural gene, that the yeast choline- and ethanolaminephosphotransferase activities are encoded by different genes, and that the CPT1 gene is nonessential for growth.     

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.     

Control of phosphatidylethanolamine metabolism in yeast: Diacylglycerol ethanolaminephosphotransferase and diacylglycerol cholinephosphotransferase are separate enzymes

Membrane preparations from Saccharomyces cerevisiae catalyze the transfer of phosphoethanolamine and phosphocholine from the cytidine dinucleotide derivatives to endogenous and exogenous 1,2-diacylglycerols. Utilizing CDP-[¹⁴C]ethanolamine and CDP-[¹⁴C]choline as isotopic substrates, diacylglycerol ethanolaminephosphotransferase (EPT) and diacylglycerol Cholinephosphotransferase (CPT) have been characterized in vitro. Both enzymes (i) require Mn²⁺; (ii) are stimulated by exogenous 1,2-diacylglycerols; and (iii) are inhibited by p-hydroxymercuribenzoate and CMP. Yeast EPT and CPT can be clearly distinguished on the basis of their different (i) pH optima; (ii) thermal sensitivities at 50 °C; (iii) concentration-dependent inhibition by CMP; and (iv) sensitivities to the hypolipidemic drug, DH-990. Reversibility experiments demonstrate that CDP-ethanolamine can be resynthesized by enzymatic reactions involving CMP and Phosphatidylethanolamine (PE) formed from the cytidine dinucleotide derivative or by the decarboxylation of phosphatidylserine (PS). Similarly, CDP-choline can be reformed by the reaction of CMP with PC synthesized from CDP-choline or by the sequential N-methylation of PE. A double-isotope experiment provides evidence that PE molecules synthesized via CDP-ethanolamine or by the decarboxylation of PS are converted to phosphatidylcholine (PC) by the methylation pathway at similar, if not identical, rates. The N-methylation of the metabolically specific pool of PE, synthesized from CDP-ethanolamine, is drastically reduced in membranes prepared from choline-grown cells. Neither EPT nor CPT appear to be induced by the addition of ethanolamine or choline, respectively, to the growth medium. However, the addition of 10 mm choline to the growth medium results in a 46% reduction in EPT activity. This change in EPT activity may be a regulatory response to lower rates of PE N-methylation in choline-grown cells.     

Glucuronosyl diacylglycerol of Pseudomonas diminuta ATCC 11568. In vitro biosynthesis from UDP-glucuronate and diacylglycerol.

The biosynthesis of glucuronosyl diacylglycerol from UDP-glucuronate and diacylglycerol is catalyzed by an enzyme found in both the 34,800 X g supernatant and particulate preparations from disrupted Pseudomonas diminuta (ATCC 11586). UDP-glucuronate served as the glucuronosyl donor and could not be replaced by glucuronic acid, glucuronate-1-phosphate, and a number of nucleotide-linked sugars. The maximum velocity was estimated to be 19 nmol of glucuronosyl diacylglycerol synthesized/h/mg of protein in the presence of the 34,800 X g particulate enzyme and 63 nmol/h/mg of protein with the 34,800 X g supernatant preparation. The apparent Km for UDP-glucuronate was 4.2 micronM for supernatant and 4.4 to 6.0 micronM for particulate preparations. The biosynthesis of glucuronosyl diacylglycerol in vitro, was strongly dependent upon exogenous diacylglycerols containing unsaturated and shorter chain fatty acids. The enzymatic activity was very heat-labile and lost about 80% of the initial rate of synthesis after preincubation for 5 min at 37 degrees. The reaction was stimulated by 14.7 mM Triton X-100 and had an optimal pH of 7.1 and an ionic strength of 0.2 M. Divalent cations were not required.